We demonstrate identifications of allotypes with 68% accuracy at the single-virus level via pattern classifications of the ionic current signatures. We also show that this discriminability becomes >95% under a binomial distribution theorem by ensembling the pulse data of >20 virions. This simple mechanism is versatile for point-of-care tests of a wide range of flu types.Rapid diagnosis of flu before symptom onsets can revolutionize our health through diminishing a risk for serious complication as well as preventing infectious disease outbreak. Sensor sensitivity and selectivity are key to accomplish this goal as the number of virus is quite small at the early stage of infection. Here we report on label-free electrical diagnostics of influenza based on nanopore analytics that distinguishes individual virions by their distinct physical features. We accomplish selective resistive-pulse sensing of single flu virus having negative surface charges in a physiological media by exploiting electroosmotic flow to filter contaminants at the Si In contrast, there is no effective way except subsidiary passive vaccination to negate a detrimental impact of the infectious pathogen ubiquitous in the environment7. A means for rapid diagnosis of the infectious disease has thus been explored as one of the strategies for preventing seasonal epidemic to possible pandemic through enabling medication at very early stage of infection10. Despite the continued progress in the performance of commercial immunosensors12, however, the sensitivity is still not high enough especially for the existing allotypes13 nor novel strains lacking antigenicity to the existing antibodies14 for diagnosis before symptom onsets., immunosensing and genetic approaches are considered as promising strategies. On the other side, while a genetic approach such as reverse transcription polymerase chain reaction16 is a versatile approach capable of identifying essentially any virus species, it relies on time consuming amplification processes with expensive facilities requiring expertise for the operation and hence not for prompt screening17. Considering that modern global society creates ever-increasing opportunities for an outbreak, it is thus of urgent importance to find sensitive sensor platforms viable for multiplex detections of the nanoscale bioparticles18.Influenza is a highly contagious respiratory disease of worldwide concern from individual to global perspectives: it annually causes millions of infections with a potential risk to serious outbreak due to the high mutability of flu virus22 for single-virus detections in a physiological environment. While it was demonstrated previously that viruses of different sizes can be discriminated by the height of resistive pulses using conventional long fluidic channels, the method is anticipated to be not applicable for distinguishing the essentially equi-sized viral nanoparticles of influenza types. The nanopores in the present study were therefore designed to have low thickness-to-diameter aspect-ratio structure28 so as to render additional sensitivity to the particle shape and surface charges whereby provide resistive pulses holding complex set of information concerning not only the nanoparticle volume but multiple physical properties of the intact viral particles. Although this would in general complicates the physical interpretation of the electrical signals wherein numerical simulations often play central roles to elucidate the electrokinetic phenomena30, we employed a machine-learning-driven pattern-analysis of the electrical signatures for rapid detection and simultaneous subtype differentiation with an ultimate sensitivity of single-particle discriminations.To this end, we herein report on a novel sensor concept capable of discriminating various types of influenza virus in label-free fashion by their distinct particle properties. We utilized a nanopore technology3N4 membrane on a Si wafer  under the applied dc voltage Vb of +0.1 V  that agrees with previous reports) to drag weakly charged biomolecules and particles into the channel .Here, special care was taken to assure that the ion Fig. . As a renel Fig. . This innel Fig.  thereby a priori expected to be a formidable task to achieve as the viruses have spherical motif of similar sizes around 100 nm irrespective of genetic variations39. In fact, we observed little difference in the resistive pulse height, a feature reflecting the volume of the pathogenic bioparticles, among the three viruses  workbench46. The algorithm randomly selects several feature parameters of an Iion spike among the predefined ten factors , where Ppre and Prec are the precision and recall calculated through the number of true-positive, false-positive, and false-negative outputs. As a result, we accomplished discrimination of A(H1N1) vs B, A(H1N1) vs A(H3N2), and A(H3N2) vs B by a single resistive pulse with accuracy of 72%, 68%, and 61%, respectively  or B infections when allowing a majority decision among the classifiers to give eventual diagnosis. Furthermore, even if the test includes allotype detections, it can be implemented at discriminability better than 78% as deduced from Prec in the most difficult case of distinguishing A(H3N2) from B  > A(H3N2) ~ B, which is consistent to the relatively low Fmeas acquired for the case of A(H3N2) vs B  ), Influenza A(H3N2) ), and Influenza B  viruses were inoculated in embryonated eggs. Specifically, we incubated chick eggs at 37 degrees Celsius and 60% humidity for 10 to 11 days. Subsequently, we inoculated one of the influenza viruses into each egg and further incubated for 3 days for multiplication. After that, we extracted chorioallantoic fluid at chorioallantois containing the multiplicated viruses. Before using for nanopore measurements, the virus solution was filtered at 450 nm  using a syringe. All the experiments using Influenza virus including inoculation, harvest, and nanopore measurements were conducted in a BSL-2 laboratory and it is confirmed by the Institutional Biosafety Committee.3N4 layer coated Si wafer was used as a substrate. We first partially removed the Si3N4 layer by a reactive ion etching (RIE) with CF4 etchant gas. The exposed Si surface was then immersed in KOH solution made by dissolving KOH pellets  in ultrapure water (Millipore Co.) at 25% concentration and heated to 100 degrees Celsius for wet etching. As a result, a deep trench was formed with a 50 nm thick Si3N4 membrane created at the bottom. On the membrane, we delineated a 300 nm diameter circle in ZEP520A resist (Zeon Co.) by an electron beam lithography. After development, the remaining resist layer was used as a mask to sculpt a 300 nm-sized nanopore by an RIE process. Finally, the substrate was kept in N,N-dimethylformamide  overnight to dissolve the residual resist followed by rinse in ethanol and acetone.A Si2 plasma treatment. On the PDMS blocks, there were microchannels formed for flowing solution through inlet and outlet holes to fill the pore. Single-virus detections were implemented by using two Ag/AgCl electrodes to measure the ionic current Iion through the nanopore. Here, a battery-driven potentiostat was used to apply the dc voltage Vb. Meanwhile, the output current was amplified by a home-made preamplifier having a bandwidth wider than 1 MHz and recorded by digitizing  and data-streaming  at 1 MHz sampling rate. Upon starting the measurements, we filled one side of a nanopore (where deep trench was formed) with ×0.25 PBS and the other side with the chorioallantoic solution containing Influenza virus of either A(H1N1), A(H3N2), or B. Clogging of a pore presumably by aggregated virus colloids was occurred in occasion. When this happened, we flown the virus solution to release the trapped particle .In prior to the ionic current measurements, two pieces of polydimethylsiloxane (PDMS) blocks were adhered on both sides of a nanopore chip immediately after activating their surface via OIion curves of every 0.5 seconds followed by subtraction of the linear component from the raw data. Then, resistive pulses were extracted by searching for the point whereat the fluctuations of the ionic current increases (decreases) above (below) 5σ to find pulse onset (offset). Furthermore, the peak regions were expanded by 0.256 ms at both sides of the onset and offset whereby defining the regions of ionic current blockade events. These processes were implemented under a home-built computer program.Resistive pulses were extracted from the ionic current traces as follows. Firstly, the moving open pore current was offsetted to zero by linearly fitting the partial Jcurr, td, r, θ, Jtime, Jnorm, Ip, Sr, S and β) depicted in Fig. hv that consists of a sequence of the average original and normalized currents at k time sections  and a time sub-vector tv defined as a sequence of the average time difference between the beginning and the peak of a pulse (tL) and that between the peak and the ending of the pulse (tR) at l current levels . Pattern analysis of ionic current spikes of A(H1N1), A(H3N2), and B was exhibited on basis of the thus acquired dataset using The Waikato Environment for Knowledge Analysis (Weka) machine learning workbench with 71 Rotation Forest ensembles where each applies a distinct base classifier such as naïve Bayes models. In this supervised learning, 142 pulses of each virus, either A(H1N1), A(H3N2), or B were used as teacher data to judge the other 48 spikes. The output was further evaluated to deduce F-Measure score Fmeas = 2PprePrec/(Ppre + Prec), where Ppre and Prec are the precision and recall calculated through TP/(TP + FP) and TP/(TP + FN), respectively, with TP, FP, FN denoting respectively the number of true-positive, false-positive, false-negative cases.Single-virus discriminations were performed by pattern-analyze individual ionic current spikes using classification algorithms of machine learning. For this, we used 60 different feature vectors which are different combinations of features and feature sub-vectors. They are 10 individual features of the ionic current pulse waveforms  algorithm. The weights which provide optimal approximation are proportional to the respective numbers of the three virus species. The precision of the estimated numbers of the three virus species are represented by ni, N are the true number of the i-th species’ pulses, its estimation wi and the total number of the virus pulses.On the other hand, statistical estimations of the respective numbers of A(H1N1), A(H3N2), and B were conducted by applying non-parametric probability density estimation of each virus in feature spaces where every feature space is formed by a pairwise combination of distinct features of the ionic current waveforms. Specifically, we chose the aforementioned Supplementary Information


We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia  10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland.Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days.The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.The online version of this article (10.1186/s12879-019-4231-9) contains supplementary material, which is available to authorized users. Viruses are the most common cause of aseptic meningitis. Depending on the season and region, a multitude of viral pathogens can produce meningitis, for example enteroviruses, tick-borne encephalitis virus, paramyxoviruses such as mumps virus, influenza virus, as well as herpesviruses. They can be detected by specific, routine PCR or serology. A rapid diagnosis of the pathogen is crucial to avoid unnecessary antibiotic or anti-herpesvirus treatment. However, in the majority of cases, the pathogen remains unknown. Metagenomic sequencing is based on unbiased detection of nucleic acids isolated from clinical samples . Hence, A 23-year-old previously healthy student from the canton of Zurich, Switzerland, presented in September 2018 with headache, nausea and photophobia for 2 days and testicular pain for one day. 10 days before onset of symptoms, he returned from a 4-day trip to Imperia . Upon admission, the patient had fever (38.0 °C) with normal heart rate (77 bpm) and blood pressure (137/82 mmHg). Physical examination revealed signs of meningeal irritation in the absence of focal neurological deficits. There were no skin abnormalities. A swollen and tender right testicle was noted. An ultrasound of the right scrotum showed a hypoechogenic inhomogeneous parenchyma and a discrete hydrocele which was compatible with epididymitis. The serum laboratory tests showed a slightly elevated C-reactive protein level of 13.0 mg/l while leucocytes were normal (8.82 G/l). A Human immunodeficiency virus (HIV)-screening test was negative.A computed tomography scan of the brain without contrast excluded brain swelling with signs of elevated intracranial pressure. A cerebrospinal fluid (CSF) examination revealed a pleocytosis of 53 cells/μl consisting primarily of lymphocytes (70%) and monocytes/macrophages (23%), normal glucose, increased lactate (3.3 mmol/l) and moderately increased total protein levels (0.821 g/l).An empirical therapy was started with intravenous acyclovir and ceftriaxone to cover bacterial and herpesvirus meningitis while awaiting CSF PCR and culture results.Treponema pallidum, and Borrelia burgdorferi. Since all tests including conventional cultures after 4 days were unremarkable, unbiased metagenomic virus sequencing of the CSF and serum was performed. Finally, fungal, bacterial and mycobacterial cultures of CSF remained negative after prolonged cultivation time.Two days later, Enterovirus, varicella zoster virus, herpes simplex virus (HSV)-1 and HSV-2 could be excluded by PCR tests and tick-borne encephalitis virus by negative IgM in CSF. Serum IgM was negative for Epstein Barr virus, Measles, Mumps, Rubella, Metagenomic sequencing of the CSF sample detected 284 reads of the species Sandfly fever Naples phlebovirus, all of them belonging to the genotype Toscana virus (TOSV). No viral reads were found in serum. Quantitative PCR specific for TOSV  performeAcyclovir was stopped after obtaining the negative herpesvirus PCR results, whereas the consulting urologist recommended to complete the course of the antibiotic treatment . After 5 days, the headache and testicular pain resolved completely, and the patient was discharged.github.com/medvir/VirMet/releases/tag/v1.1.1) [Briefly, samples were centrifuged and filtered. Total nucleic acids were extracted followed by reverse transcription with random hexamers and second strand synthesis in separate reactions for RNA and DNA genomes. For the RNA workflow, DNase treatment was included before reverse transcription. DNA and RNA preparations were kept separate for constructing sequencing libraries with the NexteraXT protocol . Libraries were sequenced on a MiSeq for 1 × 150 cycles. Reads were analyzed with a dedicated bioinformatic pipeline “VirMet” (/v1.1.1) . The rawgithub.com/medvir/SmaltAlign). In total, 4491 reads aligned and the full coding sequences could be reconstructed. However, a gap of 23 nucleotides remained in segment S at the position of the translation stop hairpin signal found in all ambisense viruses  infection in a 23-year old man. To the best of our knowledge, this is only the fourth reported case of TOSV infection in Switzerland [Sandfly fever Naples phlebovirus in the genus Phlebovirus within the family Phenuiviridae. In Eurasia and Africa, TOSV and other phleboviruses are transmitted by phlebotomine flies, which are widely distributed around the Mediterranean, in North Africa, the Middle East, India and central Asia. High seroprevalence has been recorded in humans and domestic animals in areas where sandflies are present [Toscana virus belongs to the species  present . A serop present . The inc present . Typical present , which c present –10 withoOnly three previous cases of imported TOSV cases into Switzerland have been published. These include two men, one 17-year old and one in his twenties, who were both visiting the Island of Elba , 12; andWe can’t exclude other unrecognized or unpublished cases in Switzerland since in general no routine viral metagenomics or TOSV serology is performed in patients with aseptic meningitis. Our diagnosed TOSV infection case highlights the benefits of unbiased metagenomic virus sequencing for rare or unexpected viral infections. A rapid identification of a viral etiology may have direct consequences for treatment and limit the unnecessary prescription of antibiotics. With increasing travel histories of patients, physicians should be aware of imported TOSV as the agent for viral meningitis and meningoencephalitis.Additional file 1:Figure S1. Toscana virus IgG and IgM Serology. Methods. TOSV Sequencing and PCR Primers. (PDF 4478 kb)


Varicella-zoster virus is an important human pathogen that causes varicella after primary infection and zoster after recurrence. Following primary infection, the virus remains latently for life in dorsal root and cranial nerve ganglia. Varicella and zoster are worldwide widespread diseases and may be associated with significant complications. This manuscript presents a short overview about the fundamental knowledge including the most important clinical signs, the capabilities for antiviral treatment and the spectrum of methods for laboratory diagnosis. Varicellovirus within the subfamily Alphaherpesvirinae and the family Herpesviridae. It is an enveloped DNA virus with low environmental resistance and has a size of 150–200 nm , , . For thee (UL28) . In conte (UL28) . Until nThe thymidine kinase gene of VZV has a size of 1,026 bp and is coding for 342 aa. This gene contains two conserved regions, one nucleotide (ATP)-binding site (aa 12–29) and one nucleoside (substrate)-binding site (aa 129–145). Contrary to TK of HSV-1 and HSV-2, only a low number of natural polymorphisms have been identified in the VZV TK, but it is important to differentiate each of them from resistance mutations . CompareThis gene with a size of 3,585 bp is coding for 1,195 aa. There are eight conserved regions designed to I to VII and A. Similar to the TK gene, only a small number of natural polymorphisms have been reported for DNA pol . ResistaVaricella-zoster virus-positive samples have to be considered as dangerous goods of the category B, risk group 2 to be shipped according to UN 3373 regulations. Shipment is possible at room temperature, and cooling is only recommended if samples are intended for virus isolation in cell culture . Acute VSerological VZV diagnosis is especially indicated if susceptible persons have to be identified. Because of the high rates of seroconversion, the determination of antibody status is not necessary after varicella vaccination in healthy children, adolescents, and adults in contrast to immunodeficient vaccinees and healthcare workers . In the In case of VZV infections, an antiviral treatment failure due to resistance is assumed if there are no clinical improvements detectable during the administration of the antiviral medication, mostly acyclovir within 10 –21) days . I. I35]. I50) is estimated inducing a 50% inhibition of viral replication. For the evaluation of possible resistance, a susceptible VZV reference strain has to be tested in each experiment as control. It is a crucial advantage that phenotyping allows an unambiguous interpretation of the results. However, the procedures used are time- and material-consuming as well as non-standardized. In practice, phenotypic resistance testing can only be realized if swabs can be obtained from vesicle fluids from which the virus can be isolated in cell culture. For interpretation of the results, the most common and reliable procedure for nucleoside analogs is to classify VZV strains as resistant if the mean EC50 measured is three to five times higher than the corresponding value of sensitive control strain . F. F38]. FThe author declares that he has no competing interests.This review was supported by the scientific advisory board for Antiviral Therapy of the German Association for the Control of Virus Diseases and the Society of Virology.


Invertebrateiridovirus6 (IIV6), the type species of the genus Iridovirus, including several deletions and possible recombination sites, as well as insertions of genes of non-iridoviral origin. Three isolates from vertebrate and invertebrate hosts were also used for comparative studies on pathogenicity in crickets (Gryllusbimaculatus) at 20 and 30 °C. Finally, the chameleon isolate used for the genome sequencing studies was also used in a transmission study with bearded dragons. The transmission studies showed large variability in virus replication and pathogenicity of the three tested viruses in crickets at the two temperatures. In the infection study with bearded dragons, lizards inoculated with a Liz–CrIV did not become ill, but the virus was detected in numerous tissues by qPCR and was also isolated in cell culture from several tissues. Highest viral loads were measured in the gastro-intestinal organs and in the skin. These studies demonstrate that Liz–CrIV circulates in the pet trade in Europe. This virus is capable of infecting both invertebrates and poikilothermic vertebrates, although its involvement in disease in the latter has not been proven.Invertebrate iridoviruses (IIVs), while mostly described in a wide range of invertebrate hosts, have also been repeatedly detected in diagnostic samples from poikilothermic vertebrates including reptiles and amphibians. Since iridoviruses from invertebrate and vertebrate hosts differ strongly from one another based not only on host range but also on molecular characteristics, a series of molecular studies and bioassays were performed to characterize and compare IIVs from various hosts and evaluate their ability to infect a vertebrate host. Eight IIV isolates from reptilian and orthopteran hosts collected over a period of six years were partially sequenced. Comparison of eight genome portions  showed that these were all very similar to one another and to an earlier described cricket IIV isolate, thus they were given the collective name lizard–cricket IV (Liz–CrIV). One isolate from a chameleon was also subjected to Illumina sequencing and almost the entire genomic sequence was obtained. Comparison of this longer genome sequence showed several differences to the most closely related IIV,  Iridoviridae is divided into two subfamilies, Alphairidovirinae and Betairidovirinae  quadricornis) and from the skin of a frilled lizard (Chlamydosaurus kingii) on viper heart cells (VH-2) at 28 °C quadricornis) and from the skin of a frilled lizard (Chlamydosaurus kingii)  hoehnelii) and an iguana (Iguana iguana). The chameleons were cachectic, whereas the iguana had skin lesions. Later, our laboratory isolated further IIVs from insectivorous hosts (lizards and a scorpion) and from prey crickets  . Partialrus CrIV ,32. In arus CrIV , similarcrickets . A host-An analysis of diagnostic testing for IIV in reptile, amphibian and insect samples in our lab from 2009 to 2018 shows that these viruses are still present in both captive bred insects and in reptiles and amphibians in captivity in Europe, and lengthens the list of species in which these viruses have been detected to include several new species including some snakes. It is, however, important to note that the results of some of this testing should be interpreted skeptically, since in many cases oral and/or cloacal swabs were tested. In cases in which vertebrates were fed infected insects, it is possible that virus detection in the oral cavity or even in the feces could reflect contamination from the insects rather than actual infection. On the other hand, tissues were also tested positive in several cases, including from animals that are not insectivorous, such as an Asian water monitor , No. 49 MCP gene data. Additional genetic information was necessary in order to determine whether the viruses being diagnosed in vertebrate and invertebrate animals in the pet trade in Europe were related or represented a mix of viruses, possibly including invertebrate and vertebrate specific strains. The CrIV isolate kindly sent to our laboratory by Dr. Kleespies  has identical sequences to our isolates in the four analyzed genome regions , and also in a PCR-restriction fragment length polymorphism (RFLP) analysis (author’s unpublished work). Despite the presence of several insertions, deletions and recombination sites throughout the genome of Liz–CrIV, all of our isolates studied were closely related or identical to one another in the studied genomic regions. Among our isolates there was a very low inter-isolate variance (up to 0.4%), only detected in two of the examined genes . Since the isolates studied were obtained from six different species, including lizards and arthropods over the course of six years, this indicates that CrIV has evolutionarily stable variants circulating in Europe. In this paper we referred to it as lizard–cricket iridescent virus (Liz–CrIV) or simply CrIV, since its primary host is invertebrate and the infections of vertebrate hosts appeared due to (a) single or multiple host switch(es). Which insect species the original host could be is not clear in view of the finding that the recently disclosed genome sequence of a social insect [Harpegnathos saltator), apparently harbors several mRNA and unmapped DNA sequences identical to our Liz–CrIV fragments. This suggests that this native Indian ant species is either a natural host for the CrIV or that it has been infected by the virus, e.g., during sample processing in laboratories in the USA or China [The isolates included in this study  were alll insect , Jerdon’or China ,50. ThisMCP gene sequence of Liz–CrIV is 1428 nt (coding 475 aa) long, the second longest reported in the family after IIV9  [Decapodiridovirus) [MCP gene and the closest relationship of this region to the homologous regions of polyiridoviruses, e.g., IIV9 ORF011, adjacent to the IIV9 MCP gene (ORF010). In Liz–CrIV, however, this region is a pseudogene, a non-functional homolog of IIV9 ORF011, with two stop codons in the deduced protein sequence , revealed another hypothetical recombination site (The dovirus)  and shridovirus) ,23. The dovirus) , but mucdovirus)  or IIV31sequence . The chaion site .037L) to its homolog in IIV6, the majority of the mutations and insertions in this gene clustered in close proximity, similar to the findings in the MCP gene. The structure and function of eukaryotic DNA polymerases have been characterized extensively [Despite the high similarity (97%) of the DNA polymerase gene of Liz–CrIV ? (4) Could the presence of an apparently reptile-related E3 ubiquitin protein ligase have any connection with the appearance of the Liz–CrIV in reptile hosts? Answering some of these questions will require experimental data, others can be answered using in silico analyses. There are reports in the literature proving that (regarding question 3), the same IIV genome can contain two active genes with the same enzymatic function [What selection forces enabled the emergence of the Liz–CrIV is still a puzzle. Clues that could help solve it include the non-IIV6-like ORFs , which ofunction , or  than that used in our studies, and was propagated in an insect cell line.The cricket bioassays were designed following previous studies in which extreme interassay variability was found in cricket mortality rates and in virus detection rates between trials with a single isolate . We hypoAccording to our data, the higher temperature (30 °C) does not seem to affect the ratio of patently or covertly infected crickets, although the mean survival time (MST) of the infected animals was slightly shorter at this temperature. The highest temperature at which IIV6 replication is still possible in in vitro systems is 32 °C , so it wIIV detection in lizards has been associated with a variety of clinical signs. These have included cachexia, keratoconjunctivits, hepatic hyperemia, splenomegaly, small white growths in the skeletal musculature, myxoid spindle cell sarcomas, liver lesions, apathy, CNS signs, bloody stool, hypoglycemia, sclerotic liver, stomatitis and sudden death ,35,37. SNo direct connection was found between the early death of lizard “A5”, the smallest animal included in the study, and the virus inoculation. However, the successful re-isolation of virus from every tissue and the positive nPCR results suggested viremia. This was partly supported by the qPCR results. Solitary viral particles were found in several tissues in the EM examination. However, no viral assembly sites were detected, in contrast to the findings in infected crickets . This inThe highest viral loads were found in the gastrointestinal tract, with the highest overall concentrations found in the large intestine (colon) both by virus isolation and qPCR. This was expected due to the presence of possible remnants of the infected food-crickets, despite the two weeks of vegetable diet at the end of the study. The finding of relatively high virus loads by qPCR (and virus isolation) in the proximal portions of the digestive tract of some lizards, however indicates either a great capacity of these viruses to remain within the entire digestive tract over an extended period of time, or that virus replication took place here. Other organs  also had high virus loads (by qPCR) and in some cases, the virus was also isolated from these tissues. These findings indicate that virus did spread throughout the bodies of the infected lizards.These results are in harmony with the findings in routine diagnostic lizard samples analyzed in recent years in our laboratory , includiComparing the virus loads (according to qPCR data) in the lizard tissues to those found in patently infected crickets infected with the same isolate, the values were in a three- to six-fold lower range, which could explain the difficulties encountered in finding the virus in the tissues in situ, since the sensitivity of EM is much lower than the sensitivity of e.g., virus isolation ,66.The results of these transmission studies support the hypothesis that CrIV-like viruses are able to infect vertebrates and replicate in a variety of tissues. However, when applied orally and intracoelomically, they did not induce disease. Since these viruses have been found together with other viruses in diseased lizards in several cases ,61, inclIridovirus, but shows several interesting differences in its genomic sequence, incorporating novel ORFs  with diverse homology to other viral, prokaryotic and eukaryotic sequences, and mostly unknown functions, which remain to be analyzed in subsequent research projects. Transmission studies with crickets have indicated that several factors, including environmental temperature, influence the rate of virus replication and the development of disease in invertebrate hosts. Some of these factors have not yet been elucidated. Infection studies with bearded dragons indicate that viral DNA may replicate in vertebrate hosts, but could not be associated directly with disease, or that disease development may depend on unknown circumstances.A specific strain of IIV, here called Liz–CrIV has been shown to be circulating among invertebrates in the pet trade in Europe for at least the past two decades and is also regularly found in a wide range of poikilothermic vertebrate hosts including squamate reptiles and amphibians. This virus is closely related to IIV6, the type species of the genus Evidence for the ability of these viruses to infect vertebrate hosts includes the regular detection of Liz–CrIV in diagnostic samples from vertebrates, detection of signification quantities of Liz–CrIV DNA as well as virus isolation from various tissues of bearded dragons following experimental infection, and the finding of a putative reptile-derived gene in the Liz–CrIV genome. Many details of the epidemiology of these IIVs in vertebrates as well as their possible impact on animal health require further study.


Cannas are popular ornamental plants and widely planted for the beautiful foliage and flower. Viral disease is a major threaten to canna horticulture industry. In the city of Beijing, mosaic disease in canna was frequently observed, but the associated causal agent and its biological characterization is still unknown.Sugarcane mosaic virus (SCMV) of the size ranging from 86 to 1911 nt. The complete genome of SCMV isolate (canna) was reconstructed by sequencing all cDNA clones obtained from RT-PCR and 5′\3′ RACE amplifications. SCMV-canna isolate showed to have a full RNA genome of 9579 nt in length and to share 78% nt and 85% aa sequence identities with SCMV isolates from other hosts. The phylogenetic tree constructed based on the full genome sequence of SCMV isolates allocated separately the canna-isolate in a distinct clade, indicating a new strain. Recombination analyses demonstrated that SCMV-canna isolate was a recombinant originating from a sugarcane-infecting isolate  and a maize-infecting isolate . Pathogenicity test showed SCMV-canna could cause typical symptoms of mosaic and necrosis in some tested plants with varying levels of severity but was less virulent than the isolate SCMV-BJ. Field survey showed that the virus was widely distributed.After small RNA deep sequencing, 36,776 contigs were assembled and 16 of them shared high sequence identities with the different proteins of This study identified SCMV as the major agent causing the prevalent mosaic symptom in canna plants in Beijing and its genomic and biological characterizations were further explored. All these data enriched the knowledge of the viruses infecting canna and would be helpful in effective disease management in canna. Canna indica, a member in the family Cannaceae, was listed among the most popular ornamental plants [Bean yellow mosaic virus (BYMV) and Canna yellow streak virus (CaYSV), two cucumoviruses-Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV), and a DNA virus-Canna yellow mottle virus , among which CaYSV and CaYMV were the most widely described.l plants , 2 for tl plants , 4. As oSugarcane mosaic virus subgroup with Johnsongrass mosaic virus as its closest related-virus [Alpinia in Hawaii and Piper betel L. in India [CaYSV was a newly reported potyvirus first identified in 2007 responsible for the symptoms of speckling and streaking on the leaves and had been found in UK, Belgium, Netherlands, France, Israel, USA and Russia , 6. Sequed-virus . CaYMV, ed-virus –15. Recein India , 17. Oncin India , 18. TheRNA silencing is a conserved mechanism for plants to defend against virus infection. Once infected by viruses in plants, the RNA silencing elements associated with AGO, DCL and RdRp would cleave the virus genome into small RNAs with the length ranged from 18 to 28 nts termed as virus-derived small interfering RNAs (vsiRNAs). The whole genome was the potential cleaved targets, thus the vsiRNAs were overlapped in sequences and could be assembled into contigs even complete virus genome, which supplied an alternative method for plant virus identification. With this method, many new or known plant viruses with different types of genomes were characterized –23.Sugarcane mosaic virus isolate, highly divergent from other isolates.In this study, small RNA deep sequencing analysis of canna leaves with mosaic symptom was performed to identify the causal agent associated with this disease. Sequence assembly and blast analyses showed a potyvirus present in the collected sample. The complete genome of this virus was gained with conventional RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR for both ends. The virus was identified as a A total of 21,800,471 reads was sequenced in the sample and after quality trimming 21,394,724 clean reads with the dominant size of 21-, 22- and 24 nt were obtained. The corresponding clean reads were de novo assembled, resulting in 36,776 contigs with the size ranging from 18 to 1911 nt and annotated by BLASTn and BLASTx analysis against the GenBank database. Most of the contigs were host-originated while 16 contigs ranging from 86 to 1911 nt with high levels of identity with the different proteins of SCMV by BLASTx were exclusively identified. The overlapped contigs were further manually assembled by mapping against a reference SCMV genome (AY042184). Finally the full genome sequence of SCMV-canna were finally completed with RACE PCR for both ends of the virus genome and conventional RT-PCR with overlapped specific primer pairs for the internal gaps  originating from a sugarcane-infecting isolate with the acc. no. AJ310103 as its major parent and a maize-infecting isolate with the acc. no. AJ297628 as its minor parent, indicating its origin from sugarcane-infecting isolate.This SCMV-canna isolate was 9579 nt in length, excluding the poly (A) tail at the 3′ end with the 5′ and 3′ untranslated regions (UTR) of 153 and 234 nt, respectively. Like other potyviruses, SCMV-canna contained a large open reading frame  encoding a polyprotein of 3063 amino acid (aa) residues of 346.8 kDa. BLAST analysis showed SCMV-canna shared 78% nt and 85% aa sequence identities with SCMV isolates present in the GenBank. Protein sequence analysis based on the BLASTX results , Chenopodium amaranticolor and C. quinoa. Typical mosaic symptom could be observed on all the tested maize cultivars   Stapf., Sorghum Hybrid sudan grass and Pennisetum purpureum Schum. plants while typical visible symptoms were caused by SCMV-BJ. These results were further confirmed with RT-PCR.As other SCMV isolates, SCMV-canna could not infect the plants ars Fig. a-c, f-h L.) Fig. d, which The development of next generation sequencing (NGS) technologies has greatly advanced our ability to comprehensively investigate the aetiology of viral disease . In thisPotyvirus in the family Potyviridae [Poaceae and recently SCMV was reported infecting canna in the family Cannaceae [Turnip mosaic virus was an important factor in determining the infection phenotypes in cabbage (Brassica oleracea L.) and Japanese radish (Raphanus sativus L.) [27 of Papaya ringspot virus NIa-Pro determined the host specificity for its infection of papaya [C. annuum [The ability to adapt to new hosts is an important biological property of most RNA viruses, which is particularly important for plant viruses that infect annual crop species with relatively short life and therefore need to constantly infect and adapt to new plant hosts. SCMV is a definitive member of the genus yviridae  with worannaceae . In thisivus L.) . The amif papaya . The P3 . annuum . To deteSequence comparisons showed SCMV-canna shared the highest 78 and 85% sequence identities at the nt and aa levels with specific SCMV isolates. Furthermore, SCMV-canna shared the highest nt sequence identity in the CP gene (90%) with other known isolates, while 65–80% sequence identities in other genes (The lowest in P1). This was common in plant RNA viruses with high mutation rates due to the base mismatch in replication, short generation time and large population size  and genePennisetum alopecuroides (L.) Spreng, but the symptoms were less severe than that caused by SCMV-BJ. Furthermore, the low incidence of SCMV-canna in cultivars ludan 6006 and nongda 2238 was also observed (data not shown). Early studies concluded that host range expansion was often traded off against a decreased fitness in the original host species [The symptom of mosaic leaves and dwarf plants and severe agronomical losses worldwide were often observed in sugarcane, maize, johnsongrass, millet, sorghum species, and other poaceous plants when infected by SCMV . Inocula species –42. This species . The SCM species , which wIn our study, SCMV was identified in nearly all of the canna samples with mosaic symptom. It is common that cannas are propagated via vegetative means and distributed through the frequent exchange of untested material which could disperse and increase the incidence of viral disease. Like all potyviruses, SCMV is transmitted by aphids in a non-persistent manner, thus the potential of the transmission of this isolate to maize or other host plants should be monitored as a mixed infection of different strains could change the virus virulence through recombination  which haIn conclusion, the casual agent of mosaic disease in canna was SCMV, a prevalent virus threatening the canna planting in Beijing. This isolate (SCMV-canna) shared relative low sequence identity (78% nt and 85% aa sequence identities) with SCMV isolates from other hosts and clustered into a separate branch in the phylogenetic tree. Recombinant analysis showed SCMV-canna was a naturally occurred recombinant originated from isolates infecting sugarcane and maize. The pathogenicity of SCMV-canna was relative less than the strain SCMV-BJ. This study enriched the research on viruses infecting canna and provided details on the characterization of SCMV-canna, which was helpful to the management of this disease.In October of 2014, canna plants with mosaic symptom in the leaves were observed in Beijing Botanical Garden. The symptomatic leaves from a plant were randomly collected with the Beijing Botanical Garden′s permission and the formal identification of the plant material was undertaken by the corresponding author of this article. No voucher specimens of this material were deposited. This plant was not protected or endangered species, thus no detailed permission was supplied. The samples were quickly frozen in liquid nitrogen and total RNA was extracted from the samples with TRIzol reagent  with standard procedure as recommended by the manufacturer. The quality and quantity of the RNA was assessed using a Nanodrop ND-1000  and Bio-Analyzer 2100 . Small RNAs of the length of 18–28 nt were isolated from the total RNA with 15% polyacrylamide gel for 5′ and 3′ adaptors ligation. The final ligation products were purified and reverse-transcribed for sequencing library construction. Deep sequencing was performed according to the manufacturers’ instructions using the Illumina Hiseq2000 sequencing platform.Raw Illumina sRNA reads were trimmed and cleaned by removing the sequences smaller than 16 nucleotides or longer than 30 nucleotides, low-quality tags, polyA or N tags with an in-house perl script. The resulting clean reads were assembled into contigs using the CLC Genomics Workbench which were further analyzed by BLASTn and BLASTx searches against the GenBank database. The overlapped contigs were manually assembled. Primers were then designed to amplify PCR products spanning the gaps between the various contigs and both ends of the virus genome were determined with Rapid Amplification of cDNA Ends (RACE) PCR .To understand the occurrence of viral disease in canna, a survey was conducted in 24 gardens of the Chaoyang (8), Haidian (6), Changping (7) and Yanqing 3) districts of Beijing and 64  canna plants with mosaic symptom were collected and preserved in -80 °C. Total RNAs were purified from the leaf tissues with TRIzol reagent  according to the manufacturer’s instructions. RT-PCR detection of SCMV was performed by a two-step cDNA reverse transcription with random hexanucleotide and oligo (dT) primers and the following PCR was carried out using SCMV-specific forward and reverse primers designed on sequence of our isolate ), Chenopodium , Zea , Sorghum (Sorghum sudanense (Piper) Stapf., Sorghum Hybrid sudan grass), Pennisetum , Pennisetum purpureum Schum.) were selected for pathogenicity test with three independent repeats. Inoculated plants were kept in the greenhouse  for symptom observation. All the tested plants were also inoculated with the typical SCMV isolate SCMV-BJ and PBS buffer as control.To test the infectivity of SCMV-canna, the canna leaves with mosaic symptom was homogenized in liquid nitrogen and dissolved in 0.01 mol/l phosphated buffer (pH 7.0) which was then centrifuged for separation of inoculum. Plants in the species of Additional file 1: Figure S1. Maximum Likelihood trees obtained from alignment of the complete genome nucleotide sequence (A) and amino acid sequence (B) of SCMV with 1000 bootstrap replicates. Bootstrap values are given by numbers at the relevant nodes in the topology and only bootstrap values of ≥50% were shown. Maize dwarf mosaic virus (MDMV), Pennisetum mosaic virus (PenMV), Canna yellow streak virus (CaYSV), Johnsongrass mosaic virus (JGMV) were used as outgroups.Additional file 2: Figure S2. Neighbor-joining tree obtained from alignment of the CP gene of SCMV with 1000 bootstrap replicates. Bootstrap values are given by numbers at the relevant nodes in the topology and only bootstrap values of ≥50% were shown. Maize dwarf mosaic virus (MDMV), Pennisetum mosaic virus (PenMV), Canna yellow streak virus (CaYSV), Johnsongrass mosaic virus (JGMV) were used as outgroups.Additional file 3: Table S1. Primers used for RT-PCR and Rapid Amplification of cDNA Ends (RACE) in this study.Additional file 4: Table S2. Sources of SCMV isolates used for RDP detection in this study.


POU1F1, or Pit-1) is a key transcription factor that directly regulates pituitary hormone-related genes and was considered as a candidate gene for reproduction and growth traits in goat breeding. Three important single nucleotide polymorphisms (SNPs), c.682G > T, c.723T > G and c.837T > C, were found in Shaanbei white cashmere (SBWC) goats by direct DNA sequencing, and c.876 + 110T > C was monomorphic. Further analyses showed that the c.682G > T and c.837T > C loci were associated with litter size, and c.682G > T, c.723T > G and c.837T > C strongly affected growth traits (p < 0.05). It is an interesting phenomenon for this population that individuals with the same genotypes had more offspring and that their body status was also better. Thus, we speculated that there was a positive correlation between growth and lambing ability. Besides this, female goats with H3H7 (combined from haplotype 3 and 7) diplotype (GTTT-TTTT), which was consistent with the combination of the optimal genotype of four SNPs, had a better litter size and growth status than other diplotypes. This study provides the first association analyses between the SNPs of POU1F1 and economic traits in SBWC goats, which could be considered as effective SNP markers for genomic selection in goat breeding.POU (Pit-Oct-Unc) class 1 homeobox 1  is a transcription factor that directly regulates pituitary hormone-related genes, as well as affects the reproduction and growth in mammals. Thus, POU1F1 gene was investigated as a candidate gene for litter size and growth performance in goats. In the current study, using direct DNA sequencing, c.682G > T, c.723T > G and c.837T > C loci were genotyped in Shaanbei white cashmere (SBWC) goats (n = 609), but c.876 + 110T > C was monomorphic. Besides, the c.682G > T locus was first identified by HinfI (Haemophilus influenzae Rf) restriction endonuclease. Association analysis results showed that the c.682G > T, c.837T > C loci and diplotypes were significantly associated with goat litter size (p < 0.05). The positive genotypes were GT and TT for the two SNPs, respectively, and the optimal diplotype was H3H7 (GTTT-TTTT). On the other hand, the c.682G > T, c.723T > G and c.837T > C strongly affected growth traits and body measurement indexes in SBWC goats (p < 0.05). The positive genotypes or allele of these SNPs were GT, G and TT, respectively. Additionally, the goats with H3H7 diplotype also had a greater growth status than others (p < 0.05). Here, individuals with same genotype had both a better litter size and growth traits, showing a positive correlation between these economic traits. Meanwhile, the positive genotypes of four SNPs were combined to obtain the optimal diplotype, which was also H3H7. These SNPs, especially the diplotype, could be used for the genomic selection of excellent individuals with a greater litter size and better growth status in goat breeding.POU (Pit-Oct-Unc) class 1 homeobox 1 ( As one of the earliest domesticated animals, goats play a crucial part in animal husbandry due to their extensive applications, such as in meat, cashmere, or milk production . In ordePOU1F1, initially named Pit-1) is a transcription factor belonging to the POU homeodomain family which is characterized by two highly conserved domains, the POU-specific domain and POU-Homeo domain, to bind DNA promoter regions and interact with transcriptional cofactors .,23.22,23POU1F1 polymorphisms and reproduction, the first-born litter sizes of SBWC goats were analyzed. Association analyses (p < 0.05). Briefly, female goats with the TT genotype at c.837T > C had a greater litter size than other genotypes (p = 0.001) and two types of litter size of female goats were remarkably different in each genotype distribution of c.682G > T and c.837T > C loci, respectively (p < 0.05). Thus, GT of the c.682G > T locus and TT of the c.837T > C locus were positive genotypes for litter size in SBWC goats. Moreover, it is well known that c.682G > T, c.723T > G, c.837T > C, and c.876 + 110T > C loci of goat POU1F1 were identified together in many breeds, and so the linkage relationship was analyzed among these loci in the SBWC breed. Unfortunately, there was linkage equilibrium among the four SNPs  had a better litter size and growth status than other diplotypes. This study suggests that these SNPs can be considered as effective SNP markers for genomic selection in goat breeding.The c.682G > T, c.723T > G, c.837T > C, and c.876 + 110T > C loci of


Rhizoctonia solani has a wide host range and is the causal agent of numerous crop diseases, leading to significant economic losses. To date, no cultivars showing complete resistance to R. solani have been identified and it is imperative to develop a strategy to control the spread of the disease. Fungal viruses, or mycoviruses, are widespread in all major groups of fungi and next-generation sequencing (NGS) is currently the most efficient approach for their identification. An increasing number of novel mycoviruses are being reported, including double-stranded (ds) RNA, circular single-stranded (ss) DNA, negative sense (−)ssRNA, and positive sense (+)ssRNA viruses. The majority of mycovirus infections are cryptic with no obvious symptoms on the hosts; however, some mycoviruses may alter fungal host pathogenicity resulting in hypervirulence or hypovirulence and are therefore potential biological control agents that could be used to combat fungal diseases. R. solani harbors a range of dsRNA and ssRNA viruses, either belonging to established families, such as Endornaviridae, Tymoviridae, Partitiviridae, and Narnaviridae, or unclassified, and some of them have been associated with hypervirulence or hypovirulence. Here we discuss in depth the molecular features of known viruses infecting R. solani and their potential as biological control agents.The cosmopolitan fungus  Rhizoctonia was initially described by French mycologist Augustin Pyramus de Candolle in 1815  and  and BunyRsBPLV1; . AnalysiLycoriella ingénue) as a vector for transmission [Mycovirus transmission is a significant process that needs to be addressed in any mycovirus-mediated biological control approach to alleviate fungal diseases. Specifically, it is necessary for the mycovirus to acquire some functions before being considered as a potential biological control agent, including limitation of host range to prevent the spread to undesirable hosts and the ability to establish and spread within the targeted host population . Two prismission ,106. ThiR. solani, few studies have been reported on mycovirus transmission. Successful transfection protocols were previously established for some mycoviruses, including members of the families Partitiviridae, Megabirnaviridae, Reoviridae, and Totiviridae. This approach is generally based on the use of polyethylene glycol (PEG) 4000 that promotes protoplast fusion and subsequent regeneration of the virus-transfected protoplasts and contributes substantially to the understanding of virus-host interaction and mycovirus-mediated biological control [R. solani strain GD-118 creating the derivative virus-transfected strain GD-118T [R. solani anastomosis group [Endornaviridae, which do not produce virus particles, are transmissible at high rates horizontally as well as vertically [R. solani Ra1 has the ability to be transmitted vertically via basidiospores [R. solani was transmitted horizontally through hyphal fusion, but not vertically via basidiospores [In  control . For ins GD-118T . Despite GD-118T . Purifieis group . In addirtically . For insiospores , while tiospores . Furtheriospores ,40. Moreiospores .Cryphonectria parasitica, the causal agent of chestnut blight, in Europe [Rosellinia necatrix the causative agent of a worldwide devastating disease. RnMBV1 belongs to the family Megabirnaviridae, has a bi-segmented genome and is a potential virocontrol agent since it confers hypovirulence by significantly reducing the virulence and mycelial growth of its host [Agaricus biporus caused by the ‘La France’ isometric virus (LIV) and the mushroom diseases caused by oyster mushroom isometric virus (OMIV) and oyster mushroom spherical virus (OMSV) [R. solani virus-free strain GD-118 resulted in darker mycelial pigmentation on potato dextrose agar (PDA) plates, and a reduction of mycelial growth rate, sclerotia size and numbers [R. solani with CMV does not affect the growth rate and morphology of the fungus on PDA under laboratory conditions [R. solani Rhs 1A, while sectors of the parental strain harboring the M2 dsRNA and the derivative strains showed reduced pigmentation and growth rate [Mycovirus infections are often cryptic (symptomless) but investigations focus on potential hypovirulence, a phenomenon that may be exploited in the context of sustainable biological control of fungal diseases. The prime example is Cryphonectria hypovirus 1 (CHV1), used to successfully control the plant pathogen n Europe . This din Europe . Additioits host ,69. The its host ,128. In s (OMSV) . To inves (OMSV) . For ins numbers . Further numbers . Similar numbers . In contnditions . Additiowth rate . The RNAwth rate ; the forwth rate  while thwth rate . All theR. solani are less well studied as compared to those in other fungal genera such as C. parasitica. However, a range of RNA viruses infecting R. solani was described including members of the families Barnaviridae, Benyviridae, Botourmiaviridae, Bromoviridae, Deltaflexiviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Narnaviridae, Partitiviridae, Togaviridae, and Tymoviridae, together with unclassified mycoviruses related to the orders Serpentovirales and Bunyavirales. These families include dsRNA viruses, (+)ssRNA viruses and (−)ssRNA viruses and the majority of the viruses infecting R. solani have dsRNA or (+)ssRNA genomes.Viruses infecting R. solani transcriptome following mycovirus infection. Viruses infecting R. solani are transmissible horizontally via anastomosis hyphal fusion or vertically via sporulation [R. solani CMV-free strains via hyphal fusion but failed to transmit through basidiospores [R. solani, some viruses such as CMV have no discernible effects on their host, while others such as RsPV2/GD-11 were shown to cause hypovirulence and therefore are promising biocontrol agents and should be studied extensively in the future.In addition to the discovery of novel viruses, future research on mycoviruses needs to focus on the molecular mechanisms of mycovirus–host interactions and provide a better understanding of mycovirus transmission mechanisms. Efficient mycovirus detection relies on NGS technology. NGS allows the determination of mycoviruses previously unreported and contributes considerably to the clarification of unknown molecular mechanisms of host-virus interactions since it can be used to examine in detail changes in the rulation , while srulation , whereasiospores . No spec


Are medical costs higher among older adults with a history of childhood maltreatment than among those without?In this cross-sectional study of 978 adults, using national health insurance claims data in Japan, mean annual medical costs among those who experienced any childhood maltreatment were significantly higher than costs among those who did not, even after adjusting for age and sex.In this study, childhood maltreatment was associated with additional medical costs among older adults living in Japan, which highlights the importance of primary and secondary prevention of child maltreatment. This cross-sectional study assesses whether a history of childhood maltreatment is associated with additional medical costs among older adults in Japan. Childhood maltreatment can have significant consequences on health through the life course, but its association with health care costs in later life is not widely known.To assess whether a history of childhood maltreatment is associated with additional medical costs among older adults in Japan.This population-based cross-sectional study used data from the Japan Gerontological Evaluation Study, 2013, linked with national health insurance claims data from April 2012 to March 2014 for 1 municipality that participated in the Japan Gerontological Evaluation Study, 2013. The municipality had more than 1.5 million residents, and 978 independent individuals aged 65 to 75 years were included in the analysis. Data were analyzed from October 2017 to February 2019.Childhood maltreatment, including physical abuse, emotional neglect, emotional abuse, and witnessing intimate partner violence.Mean annual medical costs between April 2012 and March 2013 and between April 2013 and March 2014.P = .007). Those who experienced emotional neglect incurred more mean medical costs than those who did not . The association of any childhood maltreatment with medical costs remained significant after controlling for age and sex . The estimated additional costs associated with childhood maltreatment would be more than ¥333 billion (US$3.1 million) per year nationwide.Among 978 independent older adults , 44 (4.5%) witnessed intimate partner violence, 19 (1.9%) were physically abused, 104 (10.6%) were emotionally neglected, and 56 (5.7%) were emotionally abused in childhood. In total, 176 older adults (18.0%) experienced at least 1 type of childhood maltreatment. Mean annual medical costs of those who experienced any childhood maltreatment were significantly higher than of those who did not (difference, ¥136 456 [US$1255]; 95% CI, ¥38 155-¥234 757 [US$351-US$2160]; In this study, childhood maltreatment was associated with additional medical costs among older adults living in Japan. This finding underlines the importance of primary and secondary prevention of child maltreatment. Furthermore, recent research using a cohort study of independent adults older than 65 years in Japan26 has shown that childhood maltreatment can lead to deteriorating health in late adulthood, including greater higher-level functional limitations and greater risk of diseases such as diabetes and hypertension.Childhood maltreatment can have long-term consequences on health through the life course28 Previous studies in the United States using health care system data30 have shown that childhood sexual abuse and/or physical abuse were associated with an increase in annual health care costs among women aged 18 to 65 years after controlling for demographic characteristics. Similar results were obtained in a Canadian study using ambulatory self-reported health care costs,31 although the amount of annual health care costs was smaller than in studies that used health care system data. Notably, these previous studies exclusively targeted women who were younger than 65 years. To our knowledge, the long-term association of childhood maltreatment with health care costs among individuals aged 65 years or older, including men, has not been examined. Moreover, no previous studies on health care costs have included the associations of childhood emotional abuse and neglect with health care costs.Therefore, experiencing childhood maltreatment may lead to additional health care costs in later life.32 However, the actual medical costs of those who experienced childhood maltreatment are still unknown because medical costs were estimated indirectly in the study. As the Japanese population is aging, elucidating the association of childhood maltreatment with health care costs among older adults is critical.In Japan, the social costs of childhood maltreatment in 2012 were reported to be ¥1.6 trillion (US$14.7 million), including direct and indirect costs.This study aimed to assess additional medical costs associated with childhood maltreatment, using the database of health insurance claims that is linked with data from the Japan Gerontological Evaluation Study (JAGES), 2013, a population-based cohort of independent adults aged 65 years or older across Japan. We also calculated additional annual medical costs for different types of childhood maltreatment .Our sample was based on participants of JAGES 2013, which was designed to investigate the social determinants of health among noninstitutionalized, functionally independent individuals aged 65 years or older. Started in 2003, JAGES is the largest cohort study of older adults in Japan, with samples from 30 municipalities in 15 prefectures.STROBE) reporting guideline.Data from JAGES 2013 were linked with the database of national health insurance claims for a city with more than 1.5 million residents, of whom 7257 were aged 75 years or younger in 2013 . Individuals with employer-sponsored health insurance or those who receive public assistance are not eligible for national health insurance. The database of health well care visits for those with national health insurance was used to distinguish between individuals who did not use national health insurance during FY2012 and FY2013  and individuals who may not have been eligible for national health insurance. Those who may not have been eligible for national health insurance, including those who have employer-sponsored health insurance and those who receive public assistance, were excluded from the sample . Medical and pharmacy claims of 5155 individuals were available for either FY2012 or FY2013. A sample of 978 individuals was used for this analysis because a randomly selected fifth of the JAGES 2013 sample was asked questions regarding adverse childhood experiences. An additional 34 participants who did not answer all 4 questions regarding childhood maltreatment were also excluded . This stMean annual medical costs, including publicly funded health care costs, for FY2012 and FY2013 were calculated from medical fee points , which were recorded in the database of national health insurance claims for city residents. These medical costs included outpatient, inpatient, and pharmaceutical costs but not dental care costs. When medical costs were available for both FY2012 and FY2013, the mean annual medical cost was used, and if only 1 FY was available, the available annual data were used as the mean annual cost for the 2 FYs.1 and modified to suit to older adults living in Japan.25 Questions regarding the 4 following types of childhood maltreatment were asked: (1) witnessing IPV, (2) experiencing physical abuse, (3) experiencing emotional neglect, and (4) experiencing emotional abuse. A participant was classified as having witnessed IPV if they answered yes to the statement, “Your father was violent with your mother.” Witnessing IPV was included as a type of childhood maltreatment in this study because IPV in front of children has been defined as child maltreatment in Japan since the Child Abuse Prevention Act was passed in 2004. A participant was classified as having experienced physical abuse if they answered yes to the statement, “(You) were hit hard by your mother/father, causing an injury.” A participant was classified as having experienced emotional neglect if they answered no to the statement “(You) felt loved by your parents.” A participant was classified as having experienced emotional abuse if they answered yes to the statement “(You) were told hurtful things or were insulted by your mother/father.” We aggregated the responses for any type of child maltreatment. In addition, the total number of maltreatment types experienced in childhood was used to assess the cumulative association of multiple types of child maltreatment with health care costs.History of maltreatment before the age of 18 years was assessed as part of the adverse childhood experiences questions in the JAGES 2013 survey, based on previous studiesAge and sex of older adults measured in the JAGES 2013 survey were used. Self-reported disease history was measured in JAGES 2013 using the following response items: high blood pressure; stroke; heart disease; diabetes; hyperlipidemia ; respiratory disease; gastrointestinal, liver, or gallbladder disease; kidney or prostate gland disease; musculoskeletal disease; traumatic injury; cancer; blood or immune system disease; depression; dementia; Parkinson disease; eye disease; ear disease; and other diseases. t tests between those who experienced any type of childhood maltreatment and those who experienced none. Considering that the distribution of medical costs was highly skewed ; the largest percentage of missing values was 1.6%, for emotional neglect. Statistical significance was set at P < .05, and all tests were 2-tailed. All analyses were conducted using Stata version 14.1 (StataCorp) from October 2017 to February 2019.We conducted P = .046) and less educated , had lower self-rated health , and had a higher prevalence of kidney or prostate gland disease  and musculoskeletal disease  than those who did not. Participants who experienced physical abuse had a higher prevalence of stroke and cancer than those who did not  (eTable 1 in the P = .02)   higher than costs among those without childhood maltreatment (P = .007) . Experiencing emotional abuse and witnessing IPV during childhood were not associated with medical costs in late adulthood ; 95% CI, −¥103 869 to ¥223 167 [−US$956 to US$1869]; P = .47; witnessing IPV: difference, −¥2604 [−US$24]; 95% CI, −¥186 186 to ¥180 979 [−US$1713 to US$1665]; P = .98).Annual medical costs in FY2012 and FY2013 ranged from 0 to ¥5 201 690 (US$47 855) . The distribution of mean annual medical costs is shown in the eFigure in the  = .007) . Mean anP = .03). Mean annual medical costs among those who experienced more than 2 types of childhood maltreatment were not significantly different from medical costs among those who experienced none.Mean annual medical costs among those who experienced 1 type of childhood maltreatment were significantly higher than costs among those who experienced none    nationwide. When kidney or prostate gland disease and musculoskeletal disease were added in model 2, any childhood maltreatment was not associated with annual medical costs .The GLMs revealed that the association of any childhood maltreatment with annual medical costs remained statistically significant after controlling for age and sex in Model 1  . Based oP = .06; model 2: AME, ¥108 215 [US$996]; SE, ¥66 279 [US$610]; 95% CI, −¥21 689 to ¥238 119 [−US$200 to US$2191]; P = .10). Mean annual medical costs among those with childhood physical abuse were not significantly different than costs among those without childhood physical abuse, although the difference was largest in model 1 . Similar to results from t tests .Emotional neglect was also not associated with mean annual medical costs . However, mean annual medical costs among those who experienced more than 2 types of childhood maltreatment did not significantly differ from medical costs among those who experienced none.Finally, the association of total number of childhood maltreatment types with mean annual medical costs among older adults was also examined in the adjusted model. Those who experienced 1 type of childhood maltreatment had significantly higher total annual medical costs than those who experienced none , given that having more adverse childhood experiences has been found to be associated with premature mortality.47Having more than 1 type of childhood maltreatment or adverse childhood experience has been reported to increase the risk of health problems31 The magnitude of the association of childhood maltreatment with medical costs was largely reduced when diseases associated with childhood maltreatment were added to our analysis model. Furthermore, our study implied that childhood physical abuse was associated with an increased risk of stroke and cancer, while emotional neglect was associated with an increased risk of kidney or prostate gland disease, which may have led to additional medical costs among independent older adults aged 65 to 74 years. This finding is in line with a previous study using the 2012 Canadian Community Health Survey data that indicated childhood physical abuse was associated with stroke, cancer, and other diseases.48 Emotional neglect has also been found to be associated with an increased risk of such diseases,49 but its association with kidney or prostate gland diseases had not been found in previous studies. Because there are a limited number of studies that examine the association of specific types of child maltreatment, especially emotional neglect, with adult diseases and different measures of diseases were used in these studies, future research using objective measures of diseases, including a wide range of diseases associated with child maltreatment, such as substance use disorders and mood disorders, is warranted to elucidate what kinds of diseases could explain the association of childhood maltreatment with increased medical costs in later life.Our study also explored whether disease history mediated the association of childhood maltreatment with medical costs in older adulthood. Our findings are consistent with previous studies, in that the long-term association of child maltreatment with health care costs seemed to be mediated by physical and mental health concerns.37 These figures suggest tremendous long-term consequences of childhood maltreatment, considering that this estimate is only for those aged 65 to 74 years who are functionally independent. Additionally, the estimate from this study is more than twice that of indirect medical costs (¥136 billion [US$1.3 billion]) estimated in the previous study in Japan.32 Our findings could contribute to more accurate estimations of the costs of child maltreatment using real health insurance claims data in Japan.Furthermore, we estimated that additional medical costs associated with childhood maltreatment were more than ¥333 billion (US$3.1 million) per year nationwide, which is approximately 0.9% of total annual medical spending  in Japan in FY2012 and FY2013 and 8.4% of total annual medical spending among those aged 65 to 74 years in FY2012 and FY2013.51 Given that another study52 suggested retrospective measures are more likely to produce false-negatives than false-positives, our findings on the association of childhood maltreatment with medical costs may be underestimated. Third, childhood sexual abuse was not assessed in our study because of the feasibility of the survey in the cultural context of Japan. However, a population-based study in Japan reported that the prevalence of childhood sexual abuse was quite low ,53 which contributed little to the underestimation of prevalence of childhood maltreatment. Furthermore, even if childhood sexual abuse were queried in surveys, previous studies in Japan55 have reported low response rates . Nonetheless, future research needs to consider how to assess childhood sexual abuse and its long-term association with health and health care costs in Japan, considering that childhood sexual abuse has been found to have significant consequences on health and health care costs later in life in other countries.31 Fourth, survival selection bias could underestimate the association of childhood maltreatment with medical costs. Fifth, the generalizability of our findings may be limited because our study only included independent adults aged 65 to 74 years in a particular city. When available, future research should include such older adults to better understand the long-term association of childhood maltreatment with medical costs using a representative sample.There are several limitations of this study. First, we might have overexcluded those who did not use national health insurance  because we could not completely distinguish between participants who were eligible for national health insurance but did not use it and those who were not eligible for national health insurance. However, a comparison of demographic information between those whose medical costs in both FY2012 and FY2013 were not available and those whose medical costs were available implies that those who were excluded from the sample seemed likely to be ineligible for national health insurance  and early intervention  for child maltreatment.


The Spanish HIV HGM BioBank is of great relevance for basic and clinical investigation, and for those groups trying to establish large networks focused on investigation on specific clinical problems. The collection of different types of samples from HIV-infected individuals is the beginning of the chain of translational investigation, starting in 2004 a prospective national HIV BioBank that expanded in 2009 a local node  for diverse pathologies and clinical networks, not only in adults but also in paediatric patients, becoming the Spanish HIV HGM BioBank. Our main objective is to find a general criteria and analytical tools to widespread its economic management to assure their sustainability and the future exploitation of the extreme high valuable biomaterial they custody.The Spanish HIV HGM BioBank was created with the aim of contributing to advance understanding of different pathologies through the transfer, management, register, processing, cryopreservation and cession of biological material from patients, always for research purposes and under conditions that guarantee its usefulness in current studies and future research that may appear as knowledge evolves. In this study, we have developed a policy for financial control and recovery costs of the Spanish HIV HGM BioBank.Actually, Spanish HIV HGM BioBank guards 413,747 vials of 46,594 samples from 16,210 donors with various prospective longitudinal study type of samples. Interestingly, more than 7907 of these samples are now used in 28 national and international investigation projects and clinical trials. One of the objectives of this study is to develop an economic plan that you get future projects, design of acceptance or rejection keys, have internal investment limits, minimum recovery needs in short/medium term, deviation detection system and a register of capital recovery by period and type of service for the Spanish HIV HGM BioBank.Our model can help BioBanks that do not have a costs recovery model to design it, as well as to detect improves and functional revisions to those experienced in this field. The development of personalized medicine is becoming a reality, creating the very best treatments thanks to research in genetic, genomic and phenotypic factors. Transferring personalized medicine to clinical practice requires research with large and varied collections of biological samples and associated clinical data. The BioBanks are research-supportive platforms that act as a link among the donors, clinics and researchers .BioBanks are complex to run from an operational and administrative point of view and must adjust to and control the intrinsic and exclusive features associated with their work, highlighting: (1) uncertainty in the use of bio-resources, maintenance of spaces for the storage, a proper control of the stocks of biomaterial, maintenance’s costs and measures to avoid accumulating material; (2) funding for BioBanks, which are mostly coming from the public administration, and difficulty in financial outlining, foreseeing, growth or control of BioBanks.HIV BioBank was created in 2004 as a national platform with the aim of contributing to advance in understanding of HIV infection. Its development increased in 2009 with the set-up of a local node of different pathologies, mostly paediatrics, always for research purposes and under conditions that guarantee its usefulness in current studies and future research that may appear as knowledge evolves (HGM node). Actually, HIV HGM BioBank receives samples from 14 HIV cohorts/studies/clinical trials and 15 collections of different pathologies in the HGM local node. Spanish HIV HGM BioBank guards 413,747 vials of 46,594 samples from 16,210 donors with various prospective longitudinal study type of samples. Since its creation more than 58,056 aliquots from 16,588 samples have been transferred in 139 national and international investigation projects and clinical trials. This activity allowed the publication of 179 articles derived of the use of its bioresources .The first step is to established of a cost analysis model. To introduce an analysis tool that settles the expenses incurred by the BioBanks –17. This(a) Analyzing the productive processes to identify the stages: (A) cost per unit of stored product; (B) cost per sample: storage and estimation of retrospective samples; (C) cost for reception and dispatch; (D) cost per order: activities carried out after donating the samples; (E) technical services: cellular, molecular and histological techniques included in the catalogue; and (F) services free of biomaterial: teaching, legal, ethics, consultancy… (b) Establishing costs targets: each stage of the productive process A, B, C, D are studied on the basis of cost/aliquot according to the type of sample. Processes E and F are based on the cost/service and/or the technique used. (c) Identifying resources that are consumed in each stage. Analysis of: reagents, perishables, courier, equipment, personnel, and other costs . (d) Assigning costs to the stages of the process. Selection of costs that are involved in each part of the process. The expenses in each stage in the Spanish HIV HGM BioBank are due to A: costs of personnel, perishables, reagents, equipment, courier and other costs. B: costs of personnel, perishables, equipment and other costs. D: costs of personnel, perishables and other costs. E: costs of personnel, reagents, perishables, equipment and other costs and F: costs of personnel, perishables and other costs. (e) Implementation and calculation of main costs: Customized budgets are prepared and registered according to our unit costs. These calculations show the price/aliquot of retrospective samples (A + B + C + D) or the investment needed to start a new collection of indeterminate (A + B + C + F) or pre-stablished (A + B + C + D + F) duration.In the Spanish HIV HGM BioBank indirect cost are personnel, equipment and other costs. In the case of personnel, the cost of the hours/product/service is assigned to each one of the productive stages. In equipment the applicable criterion is the usage time for each device, each stage of the process and redemption. Finally, the costs of electricity, water, cleaning services and telephone, is assigned to each product (cost/3 years average production), and added as “other costs” , 7.The cryopreserved material does not have donation, expiry or safety-of-use dates. The samples do not increase in value over time, but they gradually accumulate costs. Furthermore, the volume of sample transfers is very unstable, due to the fluctuating number of requests and the possibility to transfer the same sample to a single project or many different ones. For all these reasons, it is not possible to make a reliable estimation, with small variations, of the BioBank’s performance over the following year. To determine the value of the stock we carried out a study based on (1) cost/sample for the production process A and cost/sample for B in 1 year; (2) determining the number of aliquots stored in each year since 2004 until June 2018 (discounting donated samples). The global costs of 1 × 2, accounting for the time stored, enables us to know the overall value of the stocks .The Spanish HIV HGM BioBank has designed a cost recovery policy (CRP) based on different scales of discount depending on the type of service requested: Members of our hospital (HGM) and Spanish AIDS Research Network (RED RIS) − 70%, public funds − 50%, clinical trials − 35%, firms 0%.To analyze the current development of the RCP and the design of the Spanish HIV HGM BioBank’s short- and medium-term financial projections, we make a register of all the applications received that have been responded with a personalized budget. This analysis has been mandatory for all Spanish HIV HGM BioBank customers since 2016. The register includes in chronological order all the applications divided into the following categories: Collection: setting up of a cohort of prospective samples in accordance with the regulatory access. Clinical study: setting up of a collection of prospective samples for a study subsidized through a public body. Project: assignment of retrospective samples to a project. Clinical trial: assignment of retrospective/prospective samples for clinical trials. Course: teaching services. Other services: activities included in the Spanish HIV HGM BioBank catalogue.For each request processed, we recorded data on gross, incurred gross, budgeted and granted costs. Gross costs (GC): total sum of all the possible costs needed to develop a request without discounts and taking into consideration all the direct and indirect costs of the budgetary items involved. This amount reflects the complete investment that the Spanish HIV HGM BioBank needs to face for each serviced requested. It is a theoretical cost for prospective samples collections. For requests that involve the use of retrospective samples, part of the gross costs have already been paid by the Spanish HIV HGM BioBank. Only when the budget is approved by customers, costs are back paid. Incurred gross costs (IGC): these are costs resulting from the implementation of accepted budgets, without discounts and taking into consideration all the direct and indirect costs of all the budgetary items. This amount reflects the real investment that each of the requests means for the Spanish HIV HGM BioBank. Budgeted costs (BC): depending on the applicant and the funding body, the Spanish HIV HGM BioBank applies a discount policy. This budget corresponds to the amount that the Spanish HIV HGM BioBank asks for the work carried out. The budgeted costs are also theoretical until the client accepts it, and signs the MTA. Granted costs (GDC): the amount that the client/researcher finally signs for the services offered. It may not be the same as the forecasted in the budget due to negotiations with the customer or specific agreements with the Spanish HIV HGM BioBank management.The annual grouping of each block gives an idea of the real whole amounts managed by the Spanish HIV HGM BioBank, considering theoretical costs that could have been incurred, real income received, and amounts paid by the Spanish HIV HGM BioBank. The study of this structured data gives a descriptive image of the development of the Spanish HIV HGM BioBank’s funds and tries to provide a road map, find diversions and corrects them ahead.Summing up, we can group the costs in blocks and make a temporary or typological data analysis considering the ratios defined below. Average budget/request: accumulation of all the BC in a year divided by the requests analyzed. It gives an idea of the recovery average per year. Gross/budgeted costs ratio: summary of all the GC in a year divided by all the BC. It shows us the gap between the costs that may have been invested by the Spanish HIV HGM BioBank and the income expected from said activity. This ratio is useful in order to know if the discount policy designed by management fits in with the real financial needs. In this way, the discount scale for the next cycles is adjusted on the basis of information gained from previous years. Recovery from budgets (GC/GDC): This ratio comes from the sum of the theoretical GC and the real income obtained in a full financial year. It gives an idea of the success achieved in the negotiation with the client/researcher. If the collaborations are not finalized, or the gross costs are very discordant with the final budget agreement, the costs of carrying out the request and/or the negotiation process require supervision by the management and improvement. Recovery from investment (IGC/GDC) this ratio provides information on the returns from the investment. This ratio is influenced by the typology of the requests received.The annual measuring provides chronological information of the economic development from 2013 to the present, as well as global averages for each ratio. Averages and ratios give an objective measurement of the activity and efficiency in the economic management of the Spanish HIV HGM BioBank’s resources and, based on it, we can make predictions and design contingency plans for short and medium term , so we can assign to each aliquot the costs incurred from the moment of its processing up to the present day. The value of the Spanish HIV HGM BioBank’s stock in the moment of its authorization by ISCIII amounted to 62.267.822 €.The value of the Spanish HIV HGM BioBank’s stock is also contemplated in this ABC analysis. To obtain this information we calculate both the storage time of each aliquot and the costs derived from its storage in one year  costs for each task, (2) strategy of recovery and (3) set up a scale of discounts. The scope of this model is applicable in all the actions aimed to the achievement of the entity’s sustainability, accountable management and financial projections. This platform is able to know its minimum investment needs in a certain period of time since this policy was initiated. Though these data do not determine an unequivocal amount of critical resources for a future period, they allow to measure reference margins and develop preventive actions to assure the availability of economic means. Coming ahead of cutbacks, scarcity of resources, equipment acquisition and implementation of new services or prospective samples collections is essential to not endanger the future of the BioBanks –17.Costs analysis based in ABC model permitted to detect which expenditure items are particularly sensitive , 19. ThiThis is a crucial study, given that the Spanish HIV HGM BioBank activities can entail an amount of costs that exceeds the BioBank’s resources, compromising its stability. Moreover, applicants can frequently only pay once the study is ended or the funding entities grant the money. This fact results in a period of moths/years where the BioBank has to bear a sustained investment without any recovery. In this sense, a Levey–Jennings analysis allows to have an initial control over any investment, considering that Spanish HIV HGM BioBank is performing a capital invest of 3.5 times over the recovery, far from desirable 0 rate .Lack of knowledge about BioBank needs and operational procedures among researches is another important hurdle that must be overcome in the development of effective CRPs –16. In tAnother positive fact of this policy is raising awareness of the value of biomaterial , 18. TheAmong the current challenges, the heterogeneous essence of this platforms make it difficult to determine a unique model or economic framework. Moreover, there is not specific software for BioBanks’ sustainability and sometimes accounting data is short or absent. Currently, the best path to develop economic policies is the study of previous experiences and contributions among BioBanks.Nowadays, self-finance is utopic in the short-term, but Spanish HIV HGM BioBank is achieving a recovery of part of its costs. This feedback allows to improve the management of future needs in equipment acquisition, collections, prospective studies or clinical trials with a greater leverage of the BioBanks capital.A business model to maximize BioBank usage for seeking sustainable development and operation.A sustainability costs recovery policy (CRP) for financial control, including fixed and variable costs, has been developed for the BioBank.An economic plan allowing future projects including rules for acceptance or rejection, internal investment limits, minimum recovery needs in short medium term, deviation detection system and a register of capital recovery by period and type of service has been developed by our BioBank.Our model can help BioBanks that do not have a costs recovery model to design it, as well as to detect improvements and functional revisions by those experienced in this field.The future may be unclear, but what is certain is that sustainability is an issue that needs to be resolved to enable BioBanking to continue.The future may be unclear, but what is certain is that sustainability is an issue that needs to be resolved to enable BioBanking to continue.Additional file 1. List of papers directly or indirectly generated by the Spanish HIV HGM BioBank.


Increase in the evidence of global occurrence of Zika viral infection suggests that in Africa the circulation of the virus which causes 80% of asymptomatic infection could be undetected and/or overlooked. We sought to serologically detect Zika virus infection in febrile patients at Greater Accra Regional Hospital, Ghana.Of the 160 patient serum samples analyzed, 33 were found to have antibodies against Zika virus infection. Among the sero-positives 30 (91%) of the cases were anti-Zika virus IgM with the 21–30-year age group recording the highest number of 8 (26%) and 2 (7%) cases being the least for the 61 years and above age group. All sero-positive febrile patients developed at least one symptom consistent with Zika virus infection: 33 (100%) fever, 25 (76%) muscle pain, 24 (73%) joint pain, and conjunctivitis 2 (6%). Digestive symptoms recorded include 16 (49%) nausea, 12 (36%) vomiting and diarrhea 18 (55%). In addition, 28 (85%) loss of appetite, 14 (75%) rapid respiration and chest pain 15 (42%) were reported by seropositive febrile patients. Our data indicates exposure to Zika virus which suggests the possible circulation of the virus among febrile patients in Ghana with a sero-prevalence rate of 20.6%. Aedes africanus mosquito in 1948 Aedes sppted more . The higted more . The comted more . In thisThe limitation of this study was the absence of plaque neutralization reduction test and PCR for confirmation.