Chapparvovirus, a recently determined new genus in the family Parvoviridae, can infect many species of animals including bats, chickens, and pigs. Here, using viral metagenomics method, we identified a novel Chapparvovirus from feces of wild rats and designated it as rat parvovirus 2 (RPV2). The nearly complete genome of RPV2 is 4222-nt long and includes two ORFs encoding a 654-aa nonstructural protein 1 (NS1) and a 472-aa capsid protein (VP), respectively. Phylogenetic analysis over the amino acid sequence of the NS1 showed that RPV2 clustered with Eidolon helvum parvovirus 2 (EHPV2), porcine parvovirus 7 (PPV7), and turkey parvovirus 1 (TP1), forming a separate clade. Sequence analysis indicated that the NS1 protein of RPV2 shared the highest amino acid sequence identity (51 %) with that of EHPV2. According to the genetic distance-based criteria, RPV2 identified here belongs to a novel species of Chapparvovirus. Parvoviridae is divided into the subfamilies Parvovirinae and Densovirinae [Parvovirinae is divided into 8 genera: Dependoparvovirus, Copiparvovirus, Bocaparvovirus, Amdoparvovirus, Aveparvovirus, Protoparvovirus, Tetraparvovirus, and Erythroparvovirus. Recently two other genera of Marinoparvovirus and Chapparvovirus were identified. The genus of Marinoparvovirus only has one member named sesavirus which was discovered from the feces of a California sea lion pup suffering from malnutrition and pneumonia [Chapparvovirus have been detected from chicken feces samples, Eidolon helvum feces samples, and porcine multiple tissue samples including serum, rectal swab, nasal swab, and lung lavage [Chapparvovirus, as a novel genus of parvovirinae, still have unclear species. Here, using viral metagenomics and PCR amplification, we identified a novel species of Chapparvovirus from the feces of wild rats and characterized its nearly complete genome.Parvoviruses can infect many species of animals and cause illness through individual or together with other viruses –3. The vovirinae , which ineumonia . The geng lavage –8. Chappn = 15), Gaogang (n = 15), and Hailing (n = 10) from June to August in 2004. All of the wild rats were adults and the exact age was unknown.In the present study, we investigated 40 fecal samples from wild rats captured by the Chinese Center for Disease Control and Prevention in Taizhou City from three districts of Taizhou City including Taixing  and submitted to GenBank with accession no. KX272741. The near complete genome of RPV2 consists of 4222-nt and encodes two major proteins including the NS1 protein and VP protein, respectively. The NS1 protein was 654 amino acid in length possessing the ATP or GTP binding Walker loop motif 306GPSNTGKS313  motif widely presented in the other members of the subfamily Parvoviridae is lack in RPV2 VP protein [Chapparvovirus.Results indicated there were 66 sequences reads showing the best BLASTx matches to EHPV2. The nearly complete genome was then determined by filling gaps by PCR and sequenced by Sanger method. The novel  protein . The RPVParvoviridae, phylogenetic analysis over the NS1 protein of 48 members in the Parvovirinae was performed. Amino acid sequences of NS1 were aligned by ClUSTAL W, and phylogenetic trees were constructed with MEGA 6.0 software using the Neighbor-Joining statistical method based on the Jones-Taylor-Thornton matrix-based model. Result showed that RPV2 clustered with EHPV2, PPV7, and TP1, forming a separate clade . A set of nested primers designed basing on the NS1 nucleotide sequence of RPV2 was used to perform PCR screening in the 40 rat fecal samples. Primer RPV2 F1 (5′-TGGGTTCAGGCTACACAGGGTGG-3′) and RPV2 R1 (5′-CAGTCGCCCCTCTCAGGGCT-3′) were used for the first round of PCR, and RPV2 F2 (5′-AGCTGTACCCTGGGGAGAAATTGT-3′) and RPV2 R2 (5′-CGGGTTGTTGTCCTCTCGGCA-3′) for the second round, the expected length of amplified fragment was 419 bp. Our results indicated 17.5 % (7/40) of the fecal samples were positive. The PCR amplicons were T-A cloned and sequenced by Sanger method. Sequence analysis showed that the seven amplicons shared >98 % nucleotide identity, indicating a single virus strain was prevalent in the wild rats in this area.Chapparvovirus in wild rats and characterized its nearly complete genome. Phylogenetic analysis indicated that the RPV2 clustered with EHPV2, PPV7, and TP1, forming a separate clade. According to the genetic distance-based criteria, RPV2 belonged to a novel species of Chapparvovirus. Our epidemiologic data indicated RPV2 was prevalent in the wild rats in this area.In summary, we detected a novel rodent 2; PPV7, porcine parvovirus 7; RPV2, rat Chapparvovirus 2; VP, capsid proteinBLAST, basic local alignment search tool; DPBS, Dulbecco’s phosphate buffered saline; EHPV2, eidolon helvum parvovirus 2; NS1, nonstructural protein 1; ORF, open reading frame; PCR, polymerase chain reaction; PLA2, phospholipase A


Ochlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is locally common in parts of the UK where it can be a voracious feeder on people.To date there has been no evidence of mosquito-borne virus transmission of public health concern in the UK, despite the occurrence of more than 30 species of mosquito, including putative vectors of arboviruses. The saltmarsh mosquito O. detritus for three major arboviruses: dengue virus (DENV), chikungunya virus (CHIKV) and West Nile virus (WNV) using adult mosquitoes reared from wild, field-obtained immatures.Here, we assess the competence of O. detritus for either DENV or CHIKV.We demonstrate laboratory competence for WNV at 21 °C, with viral RNA detected in the mosquito’s saliva 17 days after oral inoculation. By contrast, there was no evidence of laboratory competence of O. detritus may present a potential risk for arbovirus transmission in the UK and that further investigation of its vector role in the wild is required.To our knowledge, this is the first study to demonstrate competence of a UK mosquito for WNV and confirms that  Although there have been 34 species of mosquito reported in the British Isles , includiOchlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is abundant throughout coastal regions of the British Isles, with immature mosquitoes commonly found in coastal brackish waters, particularly those prone to flooding at both the spring high tide zone and in regularly flooded saline lagoons [Ochlerotatus detritus is a multivoltine species, producing large populations following each spring flood of areas where eggs have previously been deposited awaiting saline submergence. Eggs of O. detritus can survive for over a year [Ochlerotatus detritus has a highly catholic feeding behaviour, commonly feeding on humans, birds and livestock [ lagoons , 5. Ochlr a year , with per a year . Althougr a year . Ochleroivestock , thus alivestock  and the With increasing global travel of both humans and livestock, as well as changing global climatic conditions, the geographic range of many mosquito-borne arboviruses has been increasing in recent decades. The most prominent examples of this phenomenon are West Nile virus (WNV), dengue virus (DENV) and chikungunya virus (CHIKV).WNV has expanded its range from a small area of sub-Saharan Africa to the six major continents in the last 25 years . OutbreaAe. albopictus [The incidence of new dengue cases globally is estimated to have increased 30-fold in the last 50 years , 15. Thibopictus . Furtherbopictus , 19.O. detritus are competent laboratory vectors of Japanese encephalitis virus (JEV), showing potential for transmission by the mosquito at 7 days post-infection at 23 °C [O. detritus, it is important to determine whether O. detritus is a laboratory-competent vector of the aforementioned invasive arboviruses in order to determine the risk to the UK from this potential vector. In this study, we attempted to infect O. detritus experimentally with three of the most globally important and invasive arboviruses, WNV, DENV and CHIKV, in order to determine the vector competence of this species.Previously, our group has demonstrated that field-collected at 23 °C . Given tOchlerotatus detritus immatures (fourth-instar larvae and pupae) were collected from marshland by Little Neston, Cheshire, UK . Immatures were collected using a fine scrim net and non-mosquitoes were removed from the sample using a Pasteur pipette. Immatures were reared in ambient conditions in water collected from their larval habitat until adulthood; no additional food source was provided in order to ensure the mosquitoes remained as representative of the wild population as possible. Adults were allowed to emerge and mate in 30 × 30 × 30 cm BugDorms . Control colony Ae. aegypti (New Orleans strain) (DENV and CHIKV) and Cx. quinquefasciatus (Recife strain) (WNV) were used for comparison. Colony mosquitoes were reared in an insectary at 25 °C 12:12 light:dark photoperiod and 70 % relative humidity (RH).At seven days post-emergence, female adults were removed and transferred into 1 l cylindrical polypropylene DISPO-SAFE containers, with a fine mesh covering the container opening and stored for 24 h with no access to sugar. Blood meals  containing virus (or blood only control) were provided for 3 h with an odorised feeding membrane. Unfed adults were removed from the cage, and the fed mosquitoes were incubated at 21 °C and 70 % RH for 17 days. Mortality was recorded 48 h and 17 days after feeding. 21 °C was used as this approximates a very hot summer in the south east of England. 17 days (as opposed to the standard 14 days) was used to counter the likely lengthy increase in extrinsic incubation period as a result of the relatively low experimental temperatures.6 PFU/ml; CHIKV 1 × 107 PFU/ml; DENV 1 × 107 PFU/ml, titres were limited by the available stock concentration provided by the respective institutions. Virus strains were confirmed by sequencing prior to experimentation.The virus strains were as follows: WNV NY-99, cultured at Public Health England, Porton Down, Surrey, in Vero cells; DENV Serotype 2, Bangkok Thailand; CHIKV NC/2011-568 (CHIKV_NC) cultured by the Brain Infections Group, University of Liverpool, in Vero cells. Final titres of virus in blood were as follows: WNV 2 × 10On day 17, mosquitoes were anaesthetised with FlyNap , and their saliva was extracted by inserting their proboscis into a capillary tube containing mineral oil. RNA was extracted from the expectorate using TRIzol reagent (Thermo Fisher Scientific). cDNA was generated using Superscript Vilo (Thermo Fisher Scientific).O. detritus fed with this virus. A 1/100 concentration condition was produced by serial dilution with blood, and a deactivated virus condition was produced by heating the virus (prior to adding to blood) to 70 °C for 10 min in a water bath.An additional experiment was performed for DENV using different conditions in an attempt to establish the cause of mortality in Taqman (Thermo Fisher Scientific) quantitate reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the presence of viral RNA in the samples. Primer and probe sets were as follows: WNV, sense 5′-CCA CCG GAA GTT GAG TAG ACG-3′, anti-sense 5′-TTT GGT CAC CCA GTC CTC CT-3′, probe Cy5-TGC TGC CTG CGG CTC AAC CC-BBQ, regimen 1 min at 95 °C followed by 40 cycles of 95 °C for 5 s and 60 °C for 8 s . CHIKV, O. detritus with CHIKV, although 61 % of Ae. aegypti were found to be virus-positive . However, the mortality rate of O. detritus was significantly higher at 17 days . Blood only controls from the DENV experiment below were qRT-PCR tested for CHIKV as a negative control; all individuals tested negative.No virus-positive expectorate was recorded for O. detritus had died , compared to 3 % mortality in Ae. aegypti . The experiment was repeated using four conditions: full concentration DENV (replicate 2); 1/100 dilution of virus; a heat deactivated full concentration virus; and blood only ; no significant difference was found between the two full concentration replicates . The 48 h mortality rate was significantly reduced by the virus being diluted to 1/100 concentration , as well as by the virus being heat-deactivated . There was no significant difference between 48 h mortalities with deactivated or diluted virus ; however, both deactivated and diluted virus cause significantly higher mortality at 48 h compared to the no virus (blood only) control .In the first replicate of the DENV experiment, within 48 h, 98 % of all blood-fed female O. detritus blood-fed females at 17 days were virus-positive for WNV, compared to 53 % Cx. quinquefasciatus (Table P < 0.0001). The mortality rate of Cx. quinquefasciatus was significantly higher than that of O. detritus, when infected with WNV, at both 48 h , and 17 days . Blood only controls from the DENV experiment below were qRT-PCR tested for WNV as a negative control; all individuals tested negative.Twenty-one % of the surviving O. detritus and DENV were not made as all equalled zero). There was no significant difference between the relative quantity of WNV virus recovered from the expectorate of O. detritus and Cx. quinquefasciatus .The relative quantities of virus for each experiment are shown in Fig. O. detritus was assessed for its competence for DENV, CHIKV and WNV; O. detritus was shown to be competent for only WNV. To our knowledge, this is the first demonstration that a wild-caught British mosquito is laboratory-competent for WNV and the first demonstration that O. detritus is laboratory-competent for WNV.In this study the abundant, but locally restricted, British mosquito O. detritus may be an efficient vector for WNV in the wild with the potential to sustain local transmission of WNV in the event of introduction of the virus to the UK. Furthermore, there was no significant difference between the amount of virus recovered from the expectorate of O. detritus and the known vector Cx. quinquefasciatus. Given both the catholic feeding habits of O. detritus  and the evidence presented here of its laboratory competence for WNV transmission, we suggest that the role of O. detritus as a potential risk for WNV reservoir circulation in birds, transmission to humans and transmission to horses requires consideration. WNV is considered to have been introduced into North America by either migratory birds or exotic birds transported via aeroplane [Our results raise the question as to whether eroplane . Such a eroplane , indeed,eroplane , 13. GivO. detritus for WNV, our data show that the mortality of O. detritus may be greatly increased by oral infection with DENV. We found a highly significant increase in the mortality of O. detritus compared to Ae. aegypti when using full concentration virus. However, we also showed that ‘deactivating’ the virus did not completely negate the increased mortality, and that there was no significant difference between low titre and deactivated virus. These findings could also be consistent with a contaminant causing mortality . This effect of DENV on O. detritus warrants further study; whilst it does not prove that DENV is causing the mortality, it is consistent with this theory and similar effects have been noted in some previous studies. Whilst most studies have shown no fitness costs of DENV to its host, e.g. [Ae. aegypti infected with DENV [O. detritus, it is extremely unlikely that DENV has adapted to minimize any negative fitness effects on O. detritus. Our data are therefore consistent with the prevailing theory that arboviruses adapt to minimize their effect on the longevity of their natural vectors and may have significantly greater fitness effects on non-natural vectors [In contrast to the competence of st, e.g. , there aith DENV –28. Reduith DENV , 30. The vectors .O. detritus in the low-dose DENV condition, it seems highly unlikely that O. detritus will pose a significant future risk of DENV transmission in the UK.Given both the extreme effect on longevity, together with the lack of any virus in the expectorate of O. detritus for CHIKV. Unlike the DENV infection however, CHIKV caused no significant mortality. The colony Ae. aegypti control infection did produce infectious females at 17 days at 21 °C, ruling out inactive virus. Whilst it is not known whether higher temperatures or increased time to expectorate extraction would produce infectious O. detritus, the failure to detect any CHIKV despite the long incubation period provides no evidence for risk of CHIKV transmission from this population of O. detritus.Our results also show no evidence for vector competence of O. detritus for JEV [O. detritus vectoring either DENV or CHIKV, but in contrast there is a potential risk in its role as a putative WNV vector. Ochlerotatus detritus is a nuisance mosquito species in the UK, and can be highly abundant in some coastal habitats. Given both the competence and feeding habits of O. detritus, this species may pose a credible threat for transmission of both JEV and WNV. To our knowledge, this is the first time wild UK mosquitoes have been demonstrated to be laboratory competent for WNV. However, further work is required to understand whether this laboratory competence translates into a risk of transmission in natural environments.In addition to our previous work showing competence of  for JEV , here, wNot applicable.


We have implemented BioC natural language preprocessing pipelines in two popular programming languages: C++ and Java. The current implementations interface with the well-known MedPost and Stanford natural language processing tool sets. The pipeline functionality includes sentence segmentation, tokenization, part-of-speech tagging, lemmatization and sentence parsing. These pipelines can be easily integrated along with other BioC programs into any BioC compliant text mining systems. As an application, we converted the NCBI disease corpus to BioC format, and the pipelines have successfully run on this corpus to demonstrate their functionality. Code and data can be downloaded from Database URL:http://bioc.sourceforge.net The BioCreative IV Interoperability track  addresseOur contributions to the interoperability track of the BioCreative IV challenge are BioC text-preprocessing pipelines in C++ and Java. Text preprocessing is integral to virtually all NLP systems. It processes the original text into meaningful units that contain important linguistic features before performing subsequent text mining strategies. Poor text preprocessing performance will have a detrimental effect on downstream processing. Generally, several preprocessing steps need to be performed, such as sentence segmentation, part-of-speech (POS) tagging and sentence parsing.The BioC NLP pipeline tools are an adaptation of state-of-the-art text preprocessing tools and produce corresponding text analyses in the BioC format. The tools are widely used in the target domain and have been reported to yield competitive results on biomedical texts . The int®) or Online Mendelian Inheritance in Man® (OMIM®) concepts. This resource can be used for developing machine learning methods for disease name recognition and normalization [see for example .The outputs of the BioC NLP pipeline programs provide examples of how the BioC format links the results of different tools in an interoperable and integrated fashion. This demonstrates how different programs can use and produce data in a consistent format, regardless of their implementation language. The BioC NLP pipelines are freely available to the research community and are released as open-source software that can be downloaded .The principle MedPost tool is a high accuracy POS tagger trained on a MEDLINE corpus . Using ihttp://www.sussex.ac.uk/Users/johnca/grde scription/index.html). Any initial_gr information is preserved in an infon. This is described in the cnc.key file.The C++ pipeline also includes a wrapping of the C&C dependency parser  developeAn efficient sentence segmenter, DocumentPreprocessor, is used to produce a list of sentences from a plain text. It is a creation of the Stanford NLP group using a heuristic finite-state machine that assumes the sentence ending is always signaled by a fixed set of characters. Tokenization is performed by the default rule-based tokenizer of the sentence segmenter, PTBTokenizer, before the segmenting process to divide text into a sequence of tokens. The ‘invertible’ option of the tokenizer is invoked to ensure that multiple whitespaces are reflected in token offsets so that the resulting tokens can be faithfully converted back to the original text. Sentence segmentation is then a deterministic consequence of tokenization.The MaxentTagger based on a maximum entropy model is used for POS tagging. The MaxentTagger is the default POS tagger used by the Stanford parser before parsing the text.BioLemmatizer  is used The POS-tagged sentences are then submitted to the Stanford unlexicalized natural language parser  to analyBecause of the Unicode compatibility of the integrated tools, the pipeline should work well over texts encoded in both ASCII and Unicode. The pipeline currently performs an end-to-end annotation from text tokenization to sentence parsing. However, even though sentence parsing is useful for tasks such as question answering or relation extraction, it is not often considered by tasks like named entity detection or concept recognition. In addition, because of the constituent-based parsing nature of the Stanford parser, sentence parsing accounts for most of the execution time of the pipeline. Options have been added, allowing the user to specify which stages in the pipeline should be run. This provides flexibility to run whichever stages are necessary and affordable, while avoiding unnecessary stages that require more time.We used the NCBI disease corpus to provide a use case and an evaluation of the BioC NLP tools. The NCBI disease corpus , 10 is aWe ran both C++ and Java pipelines on the BioC-formatted NCBI disease corpus, thereby enriching this resource with machine-assisted annotations and preprocessing the data ready for use by any BioC compliant application. The machine-assisted annotations consist of sentence segmentation, tokenization and POS tagging processed using both MedPost and Stanford tools, lemmatization using BioLemmatizer, parsing using both C&C and Stanford parsers, as well as abbreviation definition detection using Ab3P, Schwartz & Hearst and NatLAb algorithms .We ran the BioC NLP tools on the NCBI disease corpus and compared the outputs obtained in their native implementation versus the outputs obtained using their BioC-compatible counterparts. We found zero inconsistency in tokenization, sentence segmentation, POS tagging and parsing results during this evaluation exercise.BioC C++ and Java NLP pipelines produce easily comparable results. We performed a detailed comparison of the outputs obtained from the two pipelines on a randomly selected set of 10 PubMed abstracts. The results showed that the segmented sentences were identical. The tokens and POS tags were similar, with only expected differences. For example, MedPost and Stanford tools make different decisions on splitting tokens containing hyphens (-) or slash (/) characters. Finally, we selected a sample sentence to illustrate the dependency parsing, and results are shown in By integrating output from various parsers into a unified BioC format, our pipelines allow text mining systems to take advantage of commonalities of the structural analysis of sentences. These commonalities allow the same program to produce the visual dependency graphs in Results in a language-neutral format can be easily combined regardless of the implementation language. An example is our technical contribution to the BioNLP Shared Task 2013. Together with other groups, we were invited by the task organizers to provide automatically generated analyses of the shared task data . As the We have implemented BioC natural language preprocessing pipelines in two popular programming languages: C++ and Java. They are largely based on well-known MedPost and Stanford NLP tool sets. A benefit of BioC is the interoperability between tools written in different programming languages. With these tools, it is straightforward to use the output from Stanford tools in a C++ pipeline, or the output from MedPost in a Java pipeline. We also presented the NCBI disease corpus in the BioC format. As a test case application for the pipelines, the corpus was used as input to both the C++ and Java BioC pipelines.The BioC text preprocessing pipelines provide for automatically annotating BioC collections and can serve as starting points for more sophisticated systems. The pipelines are implemented in a flexible manner, enabling researchers to integrate other tools as needed. Alleviating the burden of interoperability challenges will encourage the exploration of novel approaches with the promise of improved text mining performance.Our BioC pipeline was used in the BioNLP 2013 shared task on event extraction to produce preprocessing results on all subtasks. These data and code are available to the public through the BioNLP shared task Web site. The shared task data were used successfully by three separate teams.http://bioc.source forge.net.BioC NLP pipeline tools and data are available to the community from the BioC Web site: Intramural Research Program of the NIH, National Library of Medicine. Funding for open access charge: Intramural Research Program of the NIH, National Library of Medicine.This research was supported by the Conflict of interest. None declared.


Autophagy is a process that mediates the degradation of cytoplasmic material, such as damaged organelles and protein aggregates, to maintain cellular homeostasis  1]. The. The1]. Autophagy is thought to be an ancient process that may have evolved to combat infection by a number of intracellular pathogens . Work frAutophagosome formation requires extensive membrane remodeling, which is also induced during the replication of positive-strand RNA viruses. Indeed, many positive strand RNA viruses including picornaviruses and flaviviruses induce the autophagic process during their replicative life cycles to generate the membranes necessary for the biogenesis of their replication organelles. In addition, a diverse array of other viruses also induce autophagy (reviewed in ), includDrosophila that limits viral replication [Host cells induce the formation of autophagosomes through a variety of mechanisms and in response to several events during the viral life cycle. During antiviral signaling, engagement of vesicular stomatitis virus by the pattern recognition receptor Toll-7 at the cell surface induces an autophagy-dependent innate immune response mediated by phosphatidylinositol 3-kinase (PI3K)-Akt-signaling in lication ,10. In alication . Some vilication . At latelication . Consistlication ,14. The lication . SeveralEnteroviruses have been extensively studied for the beneficial role that autophagy plays in their replicative life cycles. Replication of several members of the enterovirus family including poliovirus (PV) and coxsackievirus B (CVB) is enhanced by virus-induced autophagy ,17. BothIn direct contrast to enteroviruses, MeV replication benefits from autophagic flux. Despite the completion of the autophagic maturation process, MeV proteins are not targeted for degradation by autophagosome-lysosome fusion . Rather,Similar to PV, autophagy also benefits the maturation of dengue virus (DENV) particles . After rUnlike enteroviruses, DENV directly benefits from a specific autophagy-dependent process termed “lipophagy” to increase its replication. Lipophagy represents an alternative pathway of lipid metabolism and is mediated by the degradative nature of autophagic flux. During DENV infection, lipid droplets colocalize with autolysosomes, which correlates with a decrease in cellular triglycerides . ConsequA recent report has shown that human parainfluenza virus type 3 (HPIV3) infection induces the accumulation of cytoplasmic autophagosomes by the direct inhibition of autophagic flux . AutolysAccumulation of autophagosomes is also observed during influenza A virus (IAV) infection . The virHuman immunodeficiency virus-1 (HIV-1) infection also results in the subversion of the autophagic pathway to facilitate new virion formation . The HIVDespite our appreciation that many viruses utilize autophagy in a proviral manner, relatively little is known regarding the specific mechanisms by which viruses induce autophagy and/or manipulate this pathway for their gain. In addition, the term “autophagy” most commonly refers to macroautophagy, a non-selective process that mediates bulk degradation, but there are many selective forms of autophagy that are named after the degradation targets, including mitophagy (mitochondria); pexophagy (peroxisomes); aggrephagy (protein aggregates); glycophagy (glycogens); lipophagy (lipids); and, recently, ER-phagy (endoplasmic reticulum) (reviewed in ). It wil


Introduction of new vaccines in low- and lower middle-income countries has accelerated since Gavi, the Vaccine Alliance was established in 2000. This study sought to (i) estimate the costs of introducing pneumococcal conjugate vaccine, rotavirus vaccine and a second dose of measles vaccine in Zambia; and (ii) assess affordability of the new vaccines in relation to Gavi’s co-financing and eligibility policies.Data on ‘one-time’ costs of cold storage expansions, training and social mobilisation were collected from the government and development partners. A detailed economic cost study of routine immunisation based on a representative sample of 51 health facilities provided information on labour and vaccine transport costs. Gavi co-financing payments and immunisation programme costs were projected until 2022 when Zambia is expected to transition from Gavi support. The ability of Zambia to self-finance both new and traditional vaccines was assessed by comparing these with projected government health expenditures.‘One-time’ costs of introducing the three vaccines amounted to US$ 0.28 per capita. The new vaccines increased annual immunisation programme costs by 38%, resulting in economic cost per fully immunised child of US$ 102. Co-financing payments on average increased by 10% during 2008–2017, but must increase 49% annually between 2017 and 2022. In 2014, the government spent approximately 6% of its health expenditures on immunisation. Assuming no real budget increases, immunisation would account for around 10% in 2022. Vaccines represented 1% of government, non-personnel expenditures for health in 2014, and would be 6% in 2022, assuming no real budget increases.While the introduction of new vaccines is justified by expected positive health impacts, long-term affordability will be challenging in light of the current economic climate in Zambia. The government needs to both allocate more resources to the health sector and seek efficiency gains within service provision. This was switched to DTP-hepatitis B-Hib  vaccine in 2005. In 2009, a proposal was submitted for pneumococcal conjugate vaccine (PCV), rotavirus vaccine (RV) and a second dose of measles (MSD). Gavi approved PCV and MSD in 2010 and RV in 2011, following evidence of plans for cold chain expansions. However, PCV and MSD were only introduced in July 2013 and RV in November 2013. Delays were due to a measles outbreak in 2012, relocation of the Child Health Unit from the Ministry of Health to the new Ministry of Community Development, Mother and Child Health in 2011, delays in receiving the Gavi vaccine introduction grant, and delays in disbursements to subnational levels Zambia has introduced four new vaccines with Gavi support. The combined diphteria-tetanus-pertussis (DTP)-A comprehensive study on the economic and fiscal costs of Zambia’s routine immunisation services was undertaken in 2012–13, before introduction of the three new vaccines Our study objectives were to estimate the incremental costs of introducing PCV, MSD and RV and evaluate affordability after cessation of Gavi support. Although ‘one-time’ vaccine introduction costs were calculated, the primary objective was to examine the longer-term economic costs.1.1Gavi’s current eligibility criteria, established in 2011, is Gross National Income (GNI) per capita of less than US$ 1500, which is adjusted annually for inflation to remain constant in real terms. In 2015, the threshold was US$ 1580 Gavi’s co-financing policy requires countries to co-procure a portion of their new vaccines and injection equipment. MSD is exempted from co-financing, but after five years countries must take on the full costs 22.1Economic costs were estimated in 2014 values, using an exchange rate of 6.18 Zambian Kwacha for one US$ The number of fully immunised children was approximated by the number reported to receive three doses of pentavalent vaccine (penta3). With 602,000 surviving infants and 86% penta3 coverage in 2014, this was 517,720 children 2.1.1Vaccine costs were calculated by multiplying price per dose, coverage rate of the first dose, target population size, number of doses per child in the schedule and the vaccine wastage factor 2.1.23 per dose for PCV, 17.1 cm3 for RV and 2.13 cm3 for measles 3 to 138 cm3 per penta3 child Cold storage expansions were undertaken at national, provincial, district and health facility levels Power consumption for equipment was collected from WHO Product Information Sheets 2.1.3The EPIC study found that annualised costs of vehicles, vehicle maintenance and fuel comprised 12.5% of total immunisation programme costs, amounting to US$ 4.35 million in 2014 values, or US$ 8.39 per penta3 child 2.1.4As PCV and MSD were introduced concurrently, training and social mobilisation were done at the same time. Based on advice from key informants, we allocated 80% of these costs to PCV and 20% to MSD. Costs were annualised over two years, reflecting the usual frequency of training.New vaccination cards and health facility registers were printed, the Health Management Information System was modified, and a post introduction evaluation was conducted in July 2014 2.1.5Introduction of new vaccines did not lead to additional employments, but staff opportunity costs need to be accounted for. In the EPIC study, clinical staff at 51 randomly selected health facilities in nine districts were asked to allocate their working hours among all activities during one month The EPIC study took place before introduction of the new vaccines, but facility staff were asked if they thought introductions would impact operating costs, and 73% responded that they did not think so 2.2Zambia’s co-financing contributions were provided by the Gavi secretariat, and the Gavi website gave values of its vaccine and injection supplies donations Total government immunisation programme costs were the sum of systems costs, procurement of traditional vaccines and injection supplies, and government expenditure to co-procure vaccines for Gavi co-financing. The EPIC study found that immunisation systems costs amounted to US$ 31.8 million in 2011, equivalent to US$ 61 per penta3 child When assessing affordability, we compared immunisation programme costs with total government health expenditures. For 2008–2014, government health expenditures were collected from the Accountant General’s Office For 2018–2022, we assumed a nominal growth in health care expenditures of 10% per year, which was the average increase during 2014–2016, and equivalent to 5% real, annual increase with predicted inflation rates of around 5% 33.13.1.1‘One-time’, non-annualised, incremental costs amounted to US$ 4.21 million, equivalent to costs per infant in the birth cohort of US$ 0.96 for MSD, US$ 2.02 for PCV and US$ 3.58 for RV . Cold stCold storage expansions were financed by JICA (36%), ELMA Foundation (34%), Zambian government (10%), Canadian International Development Agency (9%), ARK (7%), WHO (4%), and Boston University (0.3%). Gavi, through the vaccine introduction grant, funded 64% of the social mobilisation, training and monitoring activities. The remainder was paid by the government (29%), GSK (3%), WHO (3%) and Absolute Returns for Kids (1%).3.1.2Annual costs of vaccines and injection equipment increased from US$ 6.8 million to US$ 7.0 million after introduction of MSD, to US$ 14.0 million after PCV, and to US$ 16.9 million after RV . CompareWhen ‘one-time’ costs were annualised, training and social mobilisation were the most important capital costs, each comprising 2% of total costs . Cold stThe EPIC study concluded that economic costs of routine immunisation in Zambia totalled US$ 38.2 million in 2011, equivalent to US$ 66 per penta3 child 3.23.2.1Zambia introduced pentavalent vaccine in 2005. Gavi’s first co-financing policy required countries to start co-procuring six years after introducing the first new vaccine; thus 2011 for Zambia. With GNI per capita of US$ 490 in 2005, Zambia belonged to the poorest co-financing country group, entailing flat levels of US$ 0.20 per dose for the first vaccine When Gavi’s second co-financing policy came into effect in 2012, Zambia’s GNI had increased to US$ 1,650 per capita, placing it in the intermediate group, requiring a 15% annual increase in the per dose amount. During 2012, Zambia procured the equivalent of US$ 0.27 per dose in co-financing for pentavalent vaccine and US$ 0.20 for PCV. During 2013, co-financing of RV was added, starting at US$ 0.20 per dose. During 2015, approximately 12% of pentavalent vaccines, 8% of PCV and 9% of RV were co-financed.Gavi’s new co-financing policy starts in 2016, but the first year is a grace year with the old policy still applying. Zambia is in the accelerated transition group, as its GNI per capita has exceeded the Gavi eligibility threshold for three consecutive years . Hence, Zambia is predicted to fully finance all vaccines in 2022.3.2.2During 2008–2016, per capita government health expenditures fluctuated between US$ 14 in 2009 and US$ 45 in 2013 . BetweenRoutine immunisation programme costs were US$ 52.9 million in 2014, equivalent to 0.2% of GDP. Gavi funded approximately 20% of these costs. Immunisation programme expenditures paid for by the government comprised approximately 6% of government health expenditures. Vaccine expenditures comprised 1% of government health expenditures excluding personnel . Per capAssuming a 5% annual, inflation-adjusted increase in government health expenditures during 2017–2022, immunisation programme costs are projected to be 5% of total health expenditures in 2022. When assuming 0% increase in government health expenditures, immunisation costs would account for 10% in 2022.Considering vaccine costs alone and assuming 5% real increase in government health expenditures, these will account for 3% in 2022 exclusive personnel. When assuming 0% annual increase until 2022, the proportion is 6%. For vaccine costs to remain at 1% of government health expenditures, excluding human resources, the nominal budget needs to increase by 30% each year between 2017 and 2022.4Incremental, annual systems costs of introducing the three new vaccines were US$ 4.9 million, equivalent to US$ 7.69 per child in the birth cohort and US$ 0.31 per capita. With a total of 2.8 million doses delivered, the systems costs per vaccine dose delivered of all three vaccines were US$ 1.78. The systems costs were US$ 1.33 for PCV, US$ 2.15 for RV and US$ 3.43 for MSD. Costs of simultaneous introduction of PCV, RV and MSD in Ghana in 2012 totalled US$ 2.42 per dose When including costs of vaccines, annual incremental costs of the three introductions amounted to US$ 23 per child in the birth cohort. It led to a cost increase of 38%, resulting in total immunisation programme costs of US$ 52.9 million in 2014, equivalent to US$ 102 per penta3 child. This is comparable to US$ 132 per penta3 child in Honduras, where PCV, RV and MSD have also been introduced Cold chain investments may be essential for new vaccines, and have been a major focus of cost studies of earlier new vaccine introductions Government health expenditures have fluctuated since 2008. Zambia experienced a serious economic situation in 2015. The annual economic growth rate declined from an average of 7% since 2010 to 3.4% in 2015 Immunisation is projected to comprise 5–10% of government health expenditures in 2022, compared to 6% in 2014. The proportion depends on the extent real government health expenditures increases and assuming no further deterioration in the exchange rate. Even though there are no benchmarks for optimal spending on immunisation, these proportions must be considered relatively high. Projected vaccine and injection equipment costs of 3–6% of non-personnel health spending are also higher proportions than those reported as potentially challenging in other countries Numerous studies from sub-Saharan Africa have shown substantial health impacts and cost-effectiveness of PCV, RV and MSD


We report the complete genome sequence of a lineage I peste des petits ruminants virus (E32/1969) isolated in a Senegalese laboratory in 1969. This is the earliest peste des petits ruminants virus of any lineage sequenced to date and only the second lineage I virus available in public databases. Morbillivirus within the family Paramyxoviridae , is considered an essential element in the fight for global food security and poverty alleviation. PPR is caused by the peste des petits ruminants virus (PPRV), which is a member of the genus oviridae . It is aoviridae . To dateIn March 2013 a lyophilized specimen was shipped by the Laboratoire de Virologie ISRA/LNERV, Dakar, Senegal, to the Animal Production and Health Laboratory, Vienna, for further characterization. The specimen dates back to 1969, and although the original sample from which the specimen was derived is believed to have been collected in Senegal, the sample’s exact origin is unclear. What is known, however, is that five goats were experimentally infected in June 1969 using the original sample. All of the animals died following classical PPR symptoms—namely, depression, diarrhea, respiratory difficulties, and serous and mucopurulent nasal discharges. The lungs of the dead animals were ground and passaged three times in lamb kidney cells. Aliquots (1 mL) of the infected cultures were then lyophilized in September 1969. On arrival in Vienna, the lyophilized specimen was resuspended in 1 mL of Dulbecco’s modified Eagle’s medium (high glucose medium) plus antibiotics, and 100 µL was used to infect CHS 20 cells . PhylogeThis is the earliest PPRV genome sequenced to date and only the second lineage I virus available in GenBank. The sequence provides important information to those working on the molecular evolution of this important transboundary disease.KP789375.The complete genome sequence of E32/1969 has been deposited in GenBank under the accession number


Brucea javanica (L.) Merr. (Simaroubaceae), was proven to be significantly effective in producing metabolites with anti-Tobacco mosaic virus (TMV) activities. The isolate was identified as Aspergillus tubingensis FJBJ11 based on morphological characteristics and ITS sequence. Bioassay-guided isolation led to the identification of a cycli penta-peptide, malformin A1, along with two cyclic dipeptides, cyclo (Gly-l-Pro) and cyclo (Ala-Leu). Malformin A1 showed potent inhibitory effect against the infection and replication of TMV with IC50 values of 19.7 and 45.4 μg·mL−1, as tested using local lesion assay and leaf-disc method, respectively. The results indicated the potential use of malformin A1 as a leading compound or a promising candidate of new viricide.Plant-associated microorganisms are known to produce a variety of metabolites with novel structures and interesting biological activities. An endophytic fungus FJBJ11, isolated from the plant tissue of  Tobacco mosaic virus (TMV) is one of the most well-studied plant viruses, which could infect more than 400 plant species belonging to 36 families, but until now no effective chemical treatments appeared capable of inhibiting virus replication and multiplication once it does infect host plants +.Amorphous white powder. 13C NMR  δ 169.4, 164.0, 58.1, 45.9, 44.7, 27.9, 22.1. 1H NMR  δ = 8.07 (s), 4.12 (m), 4.01 (br.s), 3.97 (br.s), 3.53 , 3.49 , 3.33 (m), 2.12 (m), 1.80 (m). ESIMS m/z 155.1 [M + H]+.Amorphous white powder. 13C NMR  δ = 169.0, 168.8, 52.6, 50.0, 42.7, 23.7, 23.0, 21.9, 19.7. 1H NMR  δ 8.13 (s), 8.10 (s), 3.86 (m), 3.76 (m), 1.81 (m), 1.60 (m), 1.46 (m), 1.26 , 1.05 (m), 0.87 , 0.85 . ESIMS m/z 185.1 [M + H]+.Amorphous white powder. 1, a cyclic penta-peptide produced from Aspergillus fungus, is proven to be a highly efficient inhibitor of Tobacco mosaic virus (TMV) infection and replication, which indicated the potential use of malformin A1 as a leading compound or a promising candidate of a new viricide.Malformin A


Carnation mottle virus (CarMV). Here, we obtained two reconstructions of CarMV at pH7 in the presence (Ca-pH7) and absence (EDTA-pH7) of calcium ions by Cryo-EM single particle analysis, which achieved 6.4 Å and 8 Å resolutions respectively. Our results showed that chelation of the calcium ions under EDTA-pH7 resulted in reduced interaction between the subunits near the center of the asymmetric unit but not overall size change of the viral particles, which indicated that the role of the calcium ions in CarMV was not predominantly for the structural preservation. Part of the genomic RNA closest to the capsid was found to be located near the center of the asymmetric unit, which might result from the interaction between genomic RNA and Lys194 residues. Together with the electrostatic potential analysis on the inner surface of the asymmetric unit, the reduced interaction near the center of the asymmetric unit under EDTA-pH7 suggested that the genome release of CarMV might be realized through the center of the asymmetric unit.Genome uncoating is a prerequisite for the successful infection of plant viruses in host plants. Thus far, little is known about the genome uncoating of the  Tombusviridae viruses are positive-strand RNA viruses with icosahedral symmetry, of which many representative structures in the genera have been determined, including Carnation mottle virus (CarMV)Tobacco necrosis virus A (TNV-A)Panicum mosaic virus (PMV)Tomato bushy stunt virus (TBSV)Turnip crinkle virus (TCV)Melon necrotic spot virus (MNSV)Cowpea mottle virus (CPMoV)Cucumber necrosis virus (CNV)Tombusviridae can, thus far, be divided into two groups: TBSV-like and TNV-like viruses. TBSV-like viruses possess obvious protruding pentamers and hexamers on the surface, while TNV-like viruses present a rather smooth surface. CarMV, TBSV, TCV, MNSV, CPMoV and CNV are all grouped into TBSV-like viruses1456745679Tombusviridae, such as the Southern bean mosaic virus (SBMV)Cowpea chlorotic mottle virus (CCMV)Carmovirus in the family Tombusviridae, the capsid of which consists of 180 capsid proteins with identical sequences1CarMV is the representative member of the genus The three-dimensional structures of CarMV were reconstructed from virus samples under Ca-pH7 and EDTA-pH7. In total, ~18, 800 particles are used for the final reconstruction under Ca-pH7 and ~10, 400 particles for the final reconstruction under EDTA-pH7. The Cryo-EM results showed that CarMV belongs to the typical T = 3 icosahedral viruses with crown-like pentamers and hexamers on the surface . The basTo compare the interaction between the subunits within one asymmetric unit, the density maps under Ca-pH7 and EDTA-pH7 were low-pass filtered at the same resolution of 8 Å, which was the lower resolution of the two reconstructions. The linking densities between the subunits within one asymmetric unit under Ca-pH7 and EDTA-pH7 were visualized . In the As a result of the limitations of the reconstruction algorithm, the structure of the genomic RNA inside the capsid could not be fully resolved. However, the electron density distribution closest to the outer shell might indicate potential interaction between the genomic RNA and capsid. In our study, the electron density distribution closest to the inner surface of the capsid was visualized, part of which was found to be located near the center of the asymmetric unit . The denThe difference between the reconstructions under Ca-pH7 and EDTA-pH7 predominantly resulted from the influence of EDTA on the structure of CarMV . In our Flock house virus (FHV) have been found to result in reduced viral stability and infectivity without changing the overall architecture of the capsidEarly biochemical experimentation has suggested that the assembly of CarMV particles does not require the presence of calcium ions16171819Genome release is a prerequisite for the successful infection of the viruses in hosts, allowing the genomic RNA to function as the template for RNA replication and protein translation, which are then used for the viral assembly2 (Ca-pH7) and 0.01 M phosphate buffer pH7 with 0.01 M EDTA (EDTA-pH7).The virus was isolated and purified from commercial stocks of carnation infected with CarMV. The sample was ground thoroughly in liquid nitrogen and then dissolved in 0.1 M phosphate buffer containing 0.1 M calcium chloride (pH7). After removing the impurities by centrifugation , a mixture of equal volumes of chloroform and butanol was added to the supernatant at 1: 4 volume and mixed for 20 min at room temperature until the stratification disappeared. The mixture was then centrifuged , resulting in the butanol phase containing virus particles in the upper layer and the chloroform phase in the lower layer. The virus particles were then precipitated by ultracentrifuge  . The resulting precipitation was resuspended and then centrifuged to remove the impurities . The final buffer conditions were 0.01 M phosphate buffer pH7 with 0.01 M CaCl2. The images were recorded on a Gatan UltraScan4000 (model 895) 16-megapixel CCD camera and the final pixel size was 1.196 Å/pixel. The defocus values ranged from −0.9~−3 μm. The EM data for the sample with the presence of EDTA were collected on an FEI Tecnai G2 TF20 at 200 kV. The images were recorded on an Eagle  4 k × 4 k CCD camera and the final pixel size was 1.35 Å/pixel.A 3 μl sample was applied to 400 mesh Quantifoil R1.2/1.3 grids  with freshly made, thin continuous carbon film, blotted for 3 s in the chamber of the Vitrobot IV at 100% humidity  and then flash-frozen in liquid ethane. The EM data for the sample with the presence of calcium ions were collected on an FEI Titan Krios at 300 kV. The total dose used for each exposure was 20 electrons/Åe2boxer.py program in EMAN2ctfit in EMAN1.9 starticos in EMAN1.9 proc3d in EMAN1.9 eotest program in EMAN1.9 Particles were interactively selected from the cryo-EM images using the https://simtk.org/home/direx/. The generated model was then docked in the density map of the asymmetric unit under EDTA-pH7 for further analysis on the linking densities in the subunit interfaces within one asymmetric unit. All the densities were normalized and visualized in ChimeraThe X-ray structure of CarMV (PDB ID: 1OPO) was downloaded and docked in the density map under Ca-pH7 using the “fit into map” module in ChimeraAccession numbers: Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with the accession numbers EMD-6468 (Ca-pH7) and EMD-6469 (EDTA-pH7).How to cite this article: Wang, C.Y. et al. Uncoating Mechanism of Carnation Mottle Virus Revealed by Cryo-EM Single Particle Analysis. Sci. Rep. 5, 14825; doi: 10.1038/srep14825 (2015).


Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV.Ebola and Marburg viruses  and urban  areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds.This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats  and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors. i.e., markers of previous contact with these viruses) in Congolese blood donors with no previous history of haemorrhagic fever. We found serologic evidence for contact with Marburg and Ebola viruses in 0.5% and 2.5% of blood donors, respectively. The circulation of Marburg virus occurs at a very low rate without any identified risk factor. In contrast, prevalence to Ebola virus was peaking at 4% in rural areas and in Pointe Noire city. Importantly, we identified that contacts with bats and birds constituted two independent environmental determinants of exposure. This study confirms that contact with Ebola virus is not infrequent in Congo and can occur in the absence of haemorrhagic fever. It highlights the requirement for further investigating the role of bats and birds in the ecological cycle of Ebola, and for determining whether asymptomatic contact with Ebola virus can provide subsequent protection against severe forms of the Ebola disease.Ebola and Marburg viruses cause haemorrhagic fevers often fatal to humans. Here, we looked for antibodies to Ebola and Marburg viruses ( Filoviridae, genera Marburgvirus and Ebolavirus) cause severe Viral Haemorrhagic Fever (VHF) in humans, with a high fatality rate in symptomatic cases [Marburg and Ebola viruses  and Ravn virus [The genus vn virus ,12. The vn virus ,14. The vn virus ,16. A sevn virus ,18.In Nigeria and DRC, seroprevalence studies identified antibodies to MARV in less than 2% of apparently healthy people selected in general population in Nigeria and amongst healthcare workers and general population in DRC ,20. In tEbolavirus includes five species: Zaire ebolavirus (Ebola virus: EBOV), Sudan ebolavirus, Taï Forest ebolavirus, Reston ebolavirus and Bundibugyo ebolavirus [Ebolavirus is primarily African in origin, with the exception of the species Reston ebolavirus which is Asian [The genus olavirus ,12. The is Asian . EBOV wais Asian  and in tis Asian ,26. Sincis Asian ,32,33,34is Asian ,36,37.versus 4.2%) [In previous seroprevalence studies, amongst 1,517 apparently healthy persons tested in five regions of the Cameroon, a positive rate of 9.7% was found with highest rates amongst Pygmies (14.5%), young adults (11.6%) and rain forest farmers (13%) . In CAR,us 4.2%) . During us 4.2%) . In a laus 4.2%) ,41. In tus 4.2%) .In Sierra Leone, in 2006–2008, among 253 febrile patients negative for Lassa fever and malaria, antibodies against EBOV and MARV were detected in respectively 8.2% et 3.2% of the samples .In this study, we present an analysis of MARV and EBOV seroprevalence amongst blood donors in the Republic of Congo in 2011 and we report associated risk factors for contact with EBOV.A MARV and EBOV seroprevalence study was performed in 2011 in the Republic of Congo, using a prospective cohort of blood donors.Field samples for the study were collected from March to July 2011, in the Republic of Congo  in urbanThis study was performed in collaboration with the Centre National de Transfusion Sanguine (CNTS) of Congo; the Virology Laboratory UMR_D 190 "Emergence des Pathologies Virales" , Marseille, France and the Virology Laboratory of Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany.Blood donors of both genders were included. The criteria for enrollment were eligibility for blood donation and provision of informed consent without specific limiting factors. The age of blood donors ranged from 18 to 65 years.Serum samples for serological analyses were collected in collaboration with the CNTS. Informed, written consent was obtained from each person enrolled in the study and the consent procedure was approved by the Congolese Research in Health Sciences Ethics Committee (N° 00000065 DGRST/CERSSA).A structured questionnaire was administered face-to-face, in the official language (French) and/or in national languages . All questionnaires were completed by the medical personnel conducting the interviews.The following data were collected: socio-demographic circumstances, domestic characteristics , environmental characteristics , travel outside the country during their lifetime, history of haemorrhagic fever .Venous blood samples were drawn using two 4 mL plain tubes which were immediately centrifuged. Sera were kept at -80°C until use. Aliquots were inactivated at 56°C for 30 min and transferred to the Virology Laboratory of the Bernhard-Nocht-Institut (BNI) in Hamburg for serological assays.Serum IgG antibodies specific for EBOV and MARV were titrated using indirect double-immunofluorescence microscopy assays which were recorded as positive if reciprocal end point titres were ≥20 .Antigens consisted of acetone-fixed Vero cells infected with Ebola virus (strain ATCC 1978) or Marburg virus (strain Popp 1967). Cultivation of the viruses was carried out in an approved and compliant BLS4 laboratory in BNI.(i) a human polyclonal antibody for the first IF with a titre at 2,560 (IgG) and (ii) a mouse monoclonal antibody with a titre at 1,280 (IgG). As positive controls for Marburg virus, we used (i) a human polyclonal antibody for the first IF with a titre at 1,280 (IgG) and (ii) a mouse monoclonal antibody with a titre at 640 (IgG).Serum samples were tested as serial twofold dilutions from 1:20 to ≥ 1:160 and antibodies were detected with a Fluorescein isothiocyanate (FITC) labelled anti-human IgG antibody-conjugate. Cell smears were counterstained with specific anti-Ebola or anti-Marburg nucleocapsid monoclonal antibodies  using a rhodamine-anti-mouse conjugate as secondary antibody . As posiThis “double immunofluorescence” protocol provides a much higher specificity than regular immunofluorescence assays, since only antibodies that detect filoviral antigens in co-localisation with a monoclonal antibody are considered.Statistical analyses were performed using the IBM SPSS statistic 21 software. Analyses aiming at analysing risk factors for seropositivity included univariate, stratified and multivariate analyses. The Fisher’s exact test was used to compare proportions in univariate analysis and the ANOVA test to compare means. The Pearson’s test was used for stratified analysis.All statistical analyses were performed at the 95% confidence level. The association between anti-EBOV IgG seropositivity and risk factors was determined by binary logistic regression analysis. Stratified analysis based on sex, age and area were performed. The significant variables in univariate analysis were entered in the multivariate model. The quality of the multivariate model was assessed with Hosmer-Lemeshow’s test.Sociodemographic characteristics are presented in Epidemiological analyses of EBOV and MARV infections were performed separately. However, the same potential risk factors for seropositivity  were assessed for both viruses.Individual titres of the MARV and EBOV-positives are presented in IgG to MARV was identified in 0.5% of the donors tested (4 in 809). Seropositivity could not be significantly associated with any of the risk factors investigated in the individual questionnaire. Antibodies to MARV were detected exclusively in male blood donors from Brazzaville who had all been in contact (touching and catching) with rodents (mice and rats), but this is a common feature in out cohort (>80% of the donors reported such contact). Three out of four were students (median age 23) and one (55yo) was an office worker. None had been in contact with (or had eaten) forest animals or bats. The titre of IgG in positive donor ranged from 80 to 160.The overall prevalence of positivity for IgG when tested against EBOV was 2.5% (20 in 809). It was 1.6% (8 in 509) in Brazzaville, 4% (3 in 75) in the rural locations  and 4% (9 in 225) in Pointe-Noire.Seropositivity in age-groups was as follows: 2.7% in 18–29yo, 2.1% in 30–39yo, 2.3% in 40–49yo, 3.2% in 50–59yo and 0.0% in >60yo, with no evidence for an increase of seroprevalence with age. In the 18–29yo age-group, higher seroprevalence was associated with touching bats (p<0.001). In the second group (30–39yo), higher seroprevalence was associated with the military profession (p = 0.04). No significant association was found in the other age-groups. No significant association was identified in a stratified analysis by occupation, including military profession.Regarding stratification by areas: in Pointe-Noire, to be a student (p = 0.002) or to have exposure to bats (p<0.001) was statistically associated with anti-EBOV IgG. Exposure to bats (p<0.001) was also found to be a risk factor in Brazzaville. Other variables  were not significantly associated with the presence of IgG, regardless of the area in which donors lived.Concerning stratification by gender , seroposIn the multivariate model , the onlWhilst the Democratic Republic of the Congo and neighbouring Angola have experienced Marburg disease outbreaks ,45, no Me.g., the number of blood donors originating from rural areas was small, all participants were older than 18 years old and 75% were males) and to the capacity to collect epidemiological information without interfering with the process of blood donation . However a body of standardised information was obtained from the questionnaires.Against this background, we conducted a seroprevalence study of Ebola and Marburg viruses amongst blood donors in the Republic of Congo, to estimate the seroprevalence of both viruses outside the epidemic period and also to identify possible risk factors. This study has some obvious limitations related to the population studied  [e.g., immunofluorescence [Importantly, our biological analyses identified antibodies specific for Ebola virus (and to a lesser extent to Marburg virus) in healthy blood donors who did not report a history of haemorrhagic fever. This confirms previous studies suggesting that filoviruses can circulate in Africa in the absence of severe clinical presentations (ections)  and thatections)  or possirescence  or ELISArescence ) and havrescence . In addirescence . Contactrescence , specifirescence , may exprescence . Altoget(i) the fact that fifteen years of investigations of African wildlife species have failed to identify such low pathogenicity strains in host species in close contact with humans and (ii) the identification of asymptomatic seroconversions following contact with symptomatic patients [It has been proposed that some populations may have been exposed to yet unidentified virus strains that have relatively low pathogenicity for humans. Monath  proposedpatients .Monath  further In the late 1980's, Gonzalez and collaborators identified low MARV seroprevalence values in Pointe Noire and Brazzaville  . In our (i) exposure to bats and (ii) bird consumption are associated independently in multivariate analysis with an increased prevalence of antibodies to EBOV is of particular relevance.Nevertheless, two remarkable aspects of our epidemiological observations deserve further discussion. On the one hand, antibodies to Ebola virus were detected in urban populations of Congolese blood donors, which should stimulate future investigations relating to asymptomatic seropositives. On the other hand, new environmental factors associated with seropositivity have been identified. In neighbouring Gabon, the seroprevalence was significantly higher in the forest regions (17.6%) than in savannah regions (10.5%) and Lakeland areas (2.7%) ,48, but The notion of bird consumption must be interpreted with caution. If birds are consumed, they will have been cooked, but bird consumption also implies contact with birds, either in the markets in Africa or when capturing them or preparing them for cooking. Whilst further studies to clarify the role of birds as a possible source of human infection are clearly justified, we prefer to refer to “exposure to birds” as a potential source of the detected seropositivity.Zaire ebolavirus nucleotide sequences were detected in six organs rodents [The possibility of exposure to birds, with supporting evidence, has never been described before as a risk factor for filovirus infection. To the best of our knowledge, birds have not been associated with EBOV or virus antigen in previous investigations . In 1979 rodents . Finally rodents .(Ficus goliath) that was full of fruits. Many birds (pigeons) were seen feeding on the figs during the day, and rodents and fruit bats were feeding on the tree during the night. It was proposed that the F. goliath could have constituted an epidemiological "hub", putting different species-including birds- in contact with one another [In 1994, an outbreak of Ebola was described in a wild chimpanzee community in the Taï National Park, Ivory Coast. Before the beginning of the outbreak, chimpanzees spent a long time in a fig tree  another .et al. [The implication of birds in the epidemiology of filoviral disease was evoked by Monath  in a hypet al.  reportedThe statistically significant identification of exposure to bats as an independent risk factor for EBOV infection is also remarkable since contact with bats has been reported as a risk factor for MARV infection ,59,60,61Our results imply that in the Republic of Congo, the circulation of Marburg virus occurs at a very low rate without any identified risk factor, but that human exposure to Ebola virus without consequent disease is not infrequent. Living in Pointe Noire or in a rural area, and having contact with bats and birds is associated with a higher risk of exposure to Ebola virus. Unfortunately, little is known about the natural history and biological properties of EBOV antibody in individuals without haemorrhagic fever. Here, we did not observe an increase of seroprevalence with age, which may suggest that, in some individuals, the antibody titre decreases and becomes undetectable with time. This would in turn imply the absence of iterative contacts with Ebola virus antigens or that of a strong antibody response following such secondary antigenic exposure.Similarly, the protection afforded by antibodies detected in blood donors against Ebola virus infection remains completely unknown. How individuals without any history of haemorrhagic fever acquire specific antibodies to EBOV and what are the biological properties of such antibodies  deserve further investigations in African populations.S1 Table(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research. Arabidopsis through both forward and reverse genetic screens.In plants and other eukaryotic organisms, small RNAs (sRNAs) have emerged as key players of RNA silencing in the regulation of various biological processes, including plant growth and development, host immunity and pathogen virulence. To date, a series of pathways beginning with different precursors and resulting in sRNAs of various sizes with dedicated functions has been elucidated in the model plant trans-acting siRNAs (ta-siRNAs), heterochromatic-associated siRNAs (hc-siRNAs), natural antisense transcript siRNAs (nat-siRNAs), and virus-activated siRNAs (vasiRNAs), which are primarily generated from diverse perfectly double-stranded RNA precursors and excised by DCL1 and its homologs  either cooperatively or redundantly  and small interfering RNAs (siRNAs). MiRNAs are generated from a primary transcript (pri-miRNA) containing a stem–loop structure and processed into 20–25 nucleotide (nt) sRNAs by RNase III endonuclease DICER-LIKE 1 (DCL1), with the exception of the DCL4-dependent miRNAs such as miR822, miR839, and miR859 . Many otundantly . sRNAs aundantly . The proundantly . Severalundantly .In regard to virus-derived small RNAs (vsiRNAs), many studies have shown in various organisms that the production of vsiRNAs is linked to antiviral immunity via silencing the viral genomic RNA. Two lines of evidences, including the biogenesis of vsiRNAs and the viral suppressors of RNA silencing (VSRs)-regulated counter-defense help us to understand the vsiRNAs-mediated antiviral RNA silencing pathway. In this review, we mainly summarize the current knowledge of the biogenesis and function of vsiRNAs in plants and discuss their potential applications as well as their future challenges in crop breeding systems.Arabidopsis and vsiRNAs profiling from various plant species. It indicates that the biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which requires the actions of various DCLs, RDRs, and AGOs. Table 1 lists the specific plant DCLs, RDRs, and AGOs known to contribute to the biogenesis of vsiRNAs.Virus-derived siRNAs, a class of exogenous sRNAs, are one of the earliest discovered sRNAs . The bioCymbidium ringspot virus (CymRSV)-infected plants led to several interesting observations: (i) vsiRNAs derived from the genomic or positive strand far outnumbered those derived from the negative strand; (ii) most vsiRNAs were concentrated in a limited number of hotspots in the CymRSV genome; and (iii) double-stranded vsiRNAs bound to VSR protein p19 appeared to contain mismatches, as they were more sensitive to RNase A digestion than fully double-stranded controls  of positive-strand RNA viruses trigger the activation of the production of vsiRNAs . If thiscontrols . These ocontrols . Severalcontrols . These rcontrols . There inscripts .Arabidopsis, the production of siRNAs from plant RNA viruses is mainly catalyzed under hierarchical action of the enzyme activities of DCL4 and DCL2 . These results were first verified with Tobacco rattle virus (TRV) infected dcl combination mutants. Specifically, TRV-specific siRNAs accumulated as discrete 21- and 24-nt species in wild-type (WT) Arabidopsis, similar to that in dcl2 mutants, while in dcl3, and dcl2/dcl3 double mutants, only 21-nt siRNAs accumulated, suggesting that DCL4 is mainly responsible for the processing of 21-nt vsiRNAs from RNA viruses -specific siRNA. Their results indicate that CMV siRNAs are produced by DCL4 in WT plants, or DCL2 and DCL3 in dcl4 mutants, which behave similar to ta-siRNAs. They also detected very low levels of 21-nt CMV-siRNAs in dcl2/dcl3/dcl4 triple mutant plants, suggesting that DCL1 could produce 21-nt CMV-siRNAs in the absence of DCL2, DCL3 and DCL4, although much less efficiently than ta-siRNAs -inoculated Arabidopsis Columbia-0 (Col-0) plants, BMV RNA accumulated higher level than that in dcl2/dcl4 double mutant plants but similar to that in dcl2 or dcl4 single mutant plants (Turnip mosaic virus (TuMV)-infected Arabidopsis, blot assays showed that the 21-nt vsiRNA from the 5′ UTR region were sensitive to loss-of-function in DCL4, but in the absence of DCL4, this region yielded siRNAs that were 22-nt in length and dependent on DCL2 (Potato virus X (PVX) in Arabidopsis was also reported (Oryza sativa endornavirus (OsEV), an endogenous dsRNA virus by increasing the accumulation of vsiRNAs, and indicated that rice DCL2 plays a major role in processing vsiRNA from OsEV , a negative-strand RNA virus, is very different in rice, Nicotiana benthamiana, and Laodelphax striatellus (insect vector). This suggests that the origins of vsiRNAs derived from different hosts are likely distinct.Members of the plant Dicer-like (DCL) protein family are the critical components of the RNA-silencing pathway that produce vsiRNAs of different lengths , DCL1 also processes some CaLCuV long dsRNAs into residual 21-nt vsiRNAs in the absence of DCL2, DCL3, and DCL4 (Cauliflower mosaic virus (CaMV), 21-, 22-, and 24-nt siRNAs from both sense and antisense polarities, coding and non-coding regions accumulated in CaMV-infected Arabidopsis . In the beginning, there was some indirect evidence to support the role of host RDRs in vsiRNAs biogenesis and antiviral defense. First, host RDRs’ activity is induced after virus infection. For instance, in tobacco mosaic virus (TMV)-infected tobacco leaves, a host polymerase RDR activity was stimulated by TMV  vsiRNA biogenesis. They found that the accumulation of RDV vsiRNAs was reduced in the OsRDR6 knockdown transgenic plants  in plants. PTGS pathways recruit suppressor of gene silencing 3 , a putative RNA trafficking protein SDE5, and the RDR6 to transform cleaved ssRNA fragments into long, perfect dsRNA precursors. Then, these are subsequently processed into 21-nt siRNAs by DCL4 and its dsRNA binding partner DRB4 or 22-nt siRNAs by DCL2 . Thus, id by TMV . Moreoved by TMV . Later, d by TMV . Among tiginally . Wang et regions , 2011. C regions . The bio regions . In rice regions  but no i regions . Recentlc plants .Arabidopsis, there are 10 AGOs, categorized into three clades, while rice has 19 AGOs classified into four clades  P14, TCV p38, CMV 2b, and RSV NS3 have been described to bind dsRNAs in a size-independent manner and therefore to block vsiRNA maturation (Rice yellow stunt virus (RYSV) P6 block secondary siRNA biogenesis by downregulating RDR6 expression or suppressing the activity of RDR1, which are critical for secondary siRNAs synthesis (Tomato yellow leaf curl virus (TYLCV) directly interact with SGS3, the cofactor of RDR6, and compete with SGS3 for dsRNA binding . Potyvirus HC-Pro, one of the best characterized viral silencing suppressors, plays multiple roles in the suppression of vsiRNAs biogenesis, such as ds-siRNA binding, blocking HEN1 methyltransferase, HEN1 binding, blocking primary siRNA biogenesis by RAV2 interaction, RDR6 downregulation. A comprehensive review on the current knowledge of the diverse VSRs and their strategies to suppress vsiRNAs biogenes is available . Notably, previous studies on miRNA-mediated antiviral RNA silencing have revealed that many molecular chaperones, such as HSP90 and GW/WG motif-containing proteins, assist in or promote the antiviral defense response . However, to date, no evidence verifies this. Additionally, there is still much that remains missing from our knowledge of vsiRNA-mediated antiviral defense due to several obstacles: (i) It remains unknown whether the majority of, if not all, vsiRNAs can load into specific AGO proteins; (ii) Even though some AGO proteins play a role in virus infection and are able to bind vsiRNAs, the RISC complex alone may not be able to access target the viral genome due to their complex secondary structures or VSR activity; (iii) At present, evidence that specific vsiRNAs can target the viral genome for inhibition of virus infection in vivo is limited. Therefore, the efficiency of antiviral RNA silencing may be determined by the accessibility of target sites  functions with DCL3 and AGO4 to induce repressive viral genome methylation against geminiviruses -derived siRNAs specifically target host CHLOROPLASTIC HEAT-SHOCK PROTEIN 90 transcripts, resulting in the albino phenotype characteristic of peach leaves infected with PLMVd (Potato spindle tuber viroid (PSTVd) variants was characterized. It targets several callose synthase genes of tomato plants , which are essential for callose formation during pathogen infection  through its F-box motif to enhance the degradation of multiple AGOs  before holo-RISC assembly. ToRSV suppressor coat protein (CP) binds to AGO1 to suppress its translational inhibitory activity and to enhance AGO1 degradation through autophagy (Sweet potato chlorotic stunt crinivirus (SPCSV), affects vsiRNA function by using a completely different strategy, in which RNase3 cleaves 21–24-nt vsiRNAs into 14 bp products and renders them inactive, thus effectively preventing the formation of antiviral RISC . Regrettably, the activity of VSRs encoded by rice viruses in suppression of vsiRNAs function is completely unknown.Current research has verified the functions of vsiRNAs in targeting viral RNAs or DNAs and, occasionally, host transcripts. In turn, many plant viruses evolved VSRs counteract the antiviral silencing process mostly through arresting the functional vsiRNAs-RISC assembly . Co-immuassembly . P25 canassembly . Polerovutophagy . RNase 3ral RISC . C2 protThough many evidences indicate that vsiRNAs play a key role in plant antiviral RNA silencing, the molecular mechanisms are elusive. Much work on the studies of biological roles of specific vsiRNAs and function mechanism of VSRs should be done in the future to further extend our understanding on the vsiRNAs function.RDV viroplasm matrix protein Pns12  led to inhibition of SCMV infection, although to varying degrees  isolate that infects sweet (Citrus sinensis Osbeck) and sour orange (C. aurantium L.) and Mexican lime (C. aurantifolia Christ.) seedlings. Caulimoviridae and Geminiviridae families by utilizing this approach, subsequently, they also reconstructed an emerging DNA virus and two viroids associated with economically important red blotch disease of grapevine to verify that vsiRNA-based deep sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants (Tobamovirus on Cicer arietinum in Europe for the first time and a viroid referring to Hop stunt viroid (NC_001351.1) was reported in chickpea based on short RNAs sequencing (Additionally, the complete genome of a certain virus can be reconstructed based on vsiRNAs derived from the virus by combining small RNA deep sequencing with variants . A new vvariants . Anotherquencing .in planta virus population for the first time. Viral RNAs and vsiRNAs both congruently portrayed the mutational landscape of the virus within the plant host (Virus-derived small interfering RNAs-based deep sequencing was also used to survey the ant host . Next geant host  and viraant host . Nowadayant host . Notablyant host , ViVaMBCant host , and Layant host , tools wIn summary, much of what we know already and what we will uncover in the future regarding plant viruses is possible by the study of vsiRNAs. Previous studies have proven that it is not only a feasible tool to control virus in crop plants by artificially expressing vsiRNAs, but also a high-throughput and cost-effective approach for reconstructing viral genomes, discovering novel virus, and studying viral ecology and populations by vsiRNAs deep sequencing experiments.With the advance of NGS technologies, it is predicted that more studies using RNA profiling of plants infected with various pathogens will help identify more vsiRNAs. However, understanding the mechanism of action of vsiRNA-mediated antiviral silencing is a big challenge for researchers. Additionally, the interactions between host plants, viruses, and the abundant various vsiRNAs are extremely complex. The question of how to determine and classify the roles of a large number of vsiRNAs in the co-evolutionary battle between hosts and viruses also remains largely unanswered. Furthermore, it is unknown whether it is a general phenomenon that the vsiRNAs are able to induce disease by targeting host endogenous genes. Until now it is unknown whether vsiRNA-mediated endogenous gene silencing is a common mechanism in virus-infected plants. Albeit great advances have been made in our knowledge of sRNA-mediated plant immunity against viruses, studies on effective antiviral drugs against plant viruses and/or approaches on improving host resistance to virus disease are still deficient. Lastly, evidence suggests a potential method for controlling viral diseases is the expression of artificial sRNAs targeting the viral genome. However, any potential risks or safety issues of using this tool in crops must be evaluated.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a result of the new SELEX method with PSA as the model target protein, the best PSA binding aptamer showed specific binding to PSA with a dissociation constant (Kd) of 0.7 nM.We demonstrate a new screening method for obtaining a prostate-specific antigen (PSA) binding aptamer based on an acoustofluidic separation (acoustophoreis) technique. Since acoustophoresis provides simultaneous washing and separation in a continuous flow mode, we efficiently obtained a PSA binding aptamer that shows high affinity without any additional washing step, which is necessary in other screening methods. In addition, next-generation sequencing (NGS) was applied to accelerate the identification of the screened ssDNA pool, improving the selecting process of the aptamer candidate based on the frequency ranking of the sequences. After the 8 Aptamer refers to short single-stranded nucleic acid that folds into a three-dimensional structure for efficient binding to their specific target with a high affinity1210111315161181920In SELEX, the most critical step is to design a system for efficient separation of the target-bound ssDNA from unspecific DNA molecules not bound to the target. A highly efficient separation can increase the affinity of the final aptamer candidates2324262730In this study, we describe a new microfluidic SELEX method based on acoustophoresis, which offers a rapid and continuous flow based process for the simultaneous separation and washing in order to enable the efficient enrichment of aptamer candidates from ssDNA libraries. No additional washing steps are needed which commonly is a requisite for other SELEX methods. By removing additional washing steps, the possibility of target loss is minimized as well as time and labor.In the principle of continuous flow based acoustophoresis, a standing wave field induces particle movement depending on its acoustic properties such as diameter, density or compressibilityWe speculated that this separation technique could be advantageous for the screening of ssDNA aptamer since efficient washing and separation is the most critical process in screening of aptamers. This speculation was supported by previous studies where acoustophoresis in a continuous flow format was applied to an efficient separation of lymphocytesNext-generation sequencing (NGS) is a high throughput sequencing technique which enables the collection of large amounts of sequence data in relatively short periods of timeIn terms of the model target protein, prostate-specific antigen (PSA), which is a well-known biomarker of prostate cancer44In this study, we demonstrate a new method for obtaining highly PSA specific aptamers assisted by continuous flow mode acoustophoresis, which encompasses a simultaneous washing and separation method without additional washing steps in combination with NGS for efficient analysis of the obtained DNA pool.As a preliminary test for recovery of beads and separation efficiency, beads suspended in PBS (0.3 mg/ml) containing Evans blue (153.8 μg/ml) were injected into the acoustophoresis separation system under the same conditions as when performing the SELEX procedure (4.93 MHz for pre-focusing and 1.99 MHz for main focusing with a flow rate of 500 μl/min).The recovery of washed beads were measured by a Coulter counter  and the bead recovery turned out to be 99.2% (the ratio of collected beads to inlet beads) and washing efficiency of Evans blue was 99.0% . Similar to the model contaminant, Evans blue, small molecules are not affected by the primary acoustic radiation force, hence the unbound ssDNA molecules and counter-proteins can be discarded through the side outlets. On the other hand, the affinity microbeads on which ssDNA is bound are driven to the center of the channel across the buffer interface and can be collected from the central outlet.14 different sequence variants with acoustophoretic setup  and binding specificity with Biacore SPR instrumentationKd value calculated to 0.7 nM. The binding of AS2 to PSA was plotted in a Michaelis-Menten curve with R2 of 0.96. The dissociation constant value of 0.7 nM is the equivalent value to the strongest reported PSA binding antibodyThe 7 selected sequences were characterized by measuring the equilibrium dissociation constants (The acoustophoretic separation (acoustophoresis) method has been reported as a simple, rapid and cost-effective separation technique49The acoustic microfluidic chip used in this study consists of two inlets and two outlets . The firIn this acoustic chip, the beads will flow through two different acoustic regions, which is the pre-focusing region of width and height dimensions 300 μm by 150 μm and the main-focusing region of dimensions 375 μm by 150 μm . FirstlyIn the subsequent main focusing region (375 μm wide), actuated at 2 MHz in a half wavelength resonance, the beads are separated from unbound ssDNA and simultaneously washed. The two lines of beads laminated along the side wall migrate into the clean buffer and merge into one stream line of beads in the center of the clean buffer flow completing the separation of beads to the central outlet and washing the unbound ssDNA to the side outlet when the acoustic force field is actuated. Because of the laminar flow, beads can be maintained in a two-dimensional stream in the main focusing channel as well as in the pre-focusing channel. The most advantageous process of the continuous flow based acoustophoretic SELEX comparing to another microfluidic-based SELEX is the simultaneous separation of target beads and washing of the unbound ssDNA due to the sheath flow and efficient acoustophoretic lateral migration of the selection beads across the sheath flow boundary. This advantage enables the elimination of additional washing steps at the same time as the separation efficiency of the bound to unbound target typically corresponds to a log 3 ratioMoreover, the continuous flow based acoustophoresis has enabled multiplex separation processSupported by these benefits, the continuous flow based acoustophoretic SELEX method was developed for screening aptamers with simultaneous separation and washing of the unbound ssDNA molecules .rd round of SELEX, the ssDNA pool was incubated with target immobilized microbeads dissolved in a mixture of counter targets, which exist most abundantly in blood, including HSA, IgG and fibrinogen for the higher specificity. After injecting this mixed solution into the acoustophoresis system, not only unbound ssDNA, but counter targets and counter targets-bound ssDNA were also eliminated. By repeating this washing procedure until the amount of bound ssDNA was saturated at 8th round  was determined by BIAcore evaluation software as well.After immobilization of PSA onto the CM5 chip, various concentrations of label free aptamer candidates, between 3 and 50 nM, dissolved in binding buffer were injected. Before the injection the aptamer dilutions were denatured at 90 °C for 10 min, followed by cooling to 4 °C for 15 min, and then incubated at 25 °C by using the thermo cycler to obtain a stable secondary ssDNA structure. The response units of aptamers bound to PSA or other counter proteins were determined and fitted with the BIAevaluation software using a 1:1 Langmuir model of binding. Each SPR curve was plotted for kinetic analysis with equation of Kd value of PSA specific aptamer AS2 turned out to be 0.7 nM, which is close to the Kd value, 0.3 nM of the antibodyDue to the efficient separation and washing based on acoustophoresis, the Kd of 0.7 nM and did not show any non-specific binding to the counter targets. This new PSA binding aptamer AS2 can be applied not only to aptamer research, but has potential to be applied in diagnosis systems for detection of prostate cancer.As a conclusion, a new microfluidic SELEX method based on microchip acoustophoresis was developed, which shows a stringent separation ability known as the most critical factor for the screening of aptamer. This acoustophoretic SELEX enables simultaneous washing and separation in a continuous flow manner. With this new SELEX method in combination with NGS, a PSA specific ssDNA aptamer (AS2) was successfully isolated. The affinity specific feature of the obtained sequence was characterized using the BIAcore SPR platform for calculation of affinity constant to PSA and specificity against its counter targets. The new PSA aptamer AS2 showed a Prostate specific antigen (PSA), human serum albumin (HSA), IgG, fibrinogen (Fib), N-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysulfosuccinimide sodium salt (NHS), and ethanolamine were purchased from Sigma-Aldrich . All other chemicals were of analytical grade and used without further treatment or purification. Double-distilled water (Millipore) was used in all experiments.The ssDNA library comprised a randomized region of 40 (N40) nucleotides flanked by two constant regions allowing primer annealing and PCR amplification .All oligodeoxynucleotides were synthesized by Integrated DNA technologies (Integrated DNA technologies (IDT), Belgium) in PAGE grade.2O) resulted in microfluidic-channels followed by dicing and anodic bonding to a 1.1 mm thick borosilicate glass lid. The width of the pre-focusing channel was 300 μm to match a wavelength resonance of 5 MHz, and the width for the main separation channel was 375 μm to match a half wavelength resonance of 2 MHz. Two piezoelectric transducing plates  were attached to the silicon chip using cyanoacrylate glue  were connected to the inlet and the outlet of the side channels of the acoustophoresis chip. The center outlet was kept open and separated beads were collected through the center outlet. The syringe pumps  controlled flow rates of each syringe. The ultrasonic standing waves were generated and transferred , to the two piezoelectric transducers. The function generators were connected to an amplifier for the pre-focus  and the main separation transducer ) respectively. The resonance frequency was 5 and 2 MHz for the pre-focusing and the main separation transducer, respectively. The temperature was controlled to 25 °C using a Peltier-controller  and the applied voltage was monitored using an oscilloscope .Target protein PSA was immobilized on p-toluenesulfonyl (tosyl)-activated magnetic beads, 2.8 μm diameter, . The tosyl groups on the magnetic beads react with primary amino groups of the protein. Briefly, 20 μg PSA dissolved in 230 μl phosphate-buffered saline (PBS) buffer was added to washed magnetic beads (5 mg). This bead-protein mixture was incubated for 24 h at 37 °C with mild rotation and the beads were blocked with 0.5% BSA for additional 1 hour according to the manufacturer’s protocol. After the coating and blocking procedure, the amount of bound PSA on the beads was calculated through measuring the amount of unbound PSA by microBCA test , and target immobilized beads were stored at 4 °C until use.14 single-stranded DNA (ssDNA) molecules containing a central random 40-nucleotide region flanked on both sides by known 18-nucleotide primer binding regions was used.A random oligonucleotide library of 1.2 × 102, and 0.02% Tween-20 in PBS, pH 7.4). Before the binding step, ssDNAs were treated by heating at 95 °C for 10 min and fast cooling at 4 °C for 15 min followed by incubation at 25 °C for 8 min as a pre-requisite for stable secondary ssDNA structure. Then the SELEX process was initiated by first incubation of PSA coated beads with 1.2 × 1014 of random ssDNAs from the library in a total reaction volume of 500 μl of binding buffer for 30 min. The ssDNAs bound to the target were separated from unbound or weakly bound ssDNAs by washing with acoustophoretic separation. After collecting the separated beads, the bound ssDNAs were eluted by suspending the beads containing ssDNAs bound to PSA in 200 μl of elution buffer . The elution process was performed by heating the suspension at 95 °C for 10 min with mild shaking of the beads bound to target oligonucleotides. The eluted PSA-bound ssDNA fraction was purified by ethanol precipitation and amplified by polymerase chain reaction (PCR) using a fluorescent forward primer and an unlabeled reverse primer.Binding reactions were performed in binding buffer (2 mM MgClA double-stranded DNA (dsDNA) product was obtained after PCR using the following program: initial denaturation for 5 min at 94 °C and 10 cycles for 1 min at 94 °C, 1 min at 53 °C, 1 min at 72 °C, and extension for 10 min at 72 °C. The PCR product was subjected to denaturing polyacrylamide gel electrophoresis to isolate ssDNA fractions containing potential aptamer candidates from the PCR product . The flurd round of enrichment was incubated with bare beads first. The ssDNA pool that did not bind to the bare beads was separated using magnetic separation and then subjected to the mixture of PSA coated beads and counter targets including HSA, IgG and fibrinogen, which are freeform in the buffer condition. Counter selection was conducted after the 3rd round until enrichment was finished.In addition, to avoid non-specific binding, the aptamer pool was subjected to counter selection steps against bare magnetic beads and counter targets including HSA, fibrinogen and IgG, which are known to be the most 3 abundant proteins in blood. For counter selection, the ssDNA pool from the 3NGS was performed using Illumina’s Mi-seq . The selected pools were PCR amplified to dsDNAs with an optimum cycle by primers , and then purified by MinElute Kit . The purified dsDNAs were amplified using sequencing primers (with 6 bp barcodes) by PCR and purified before sequencing. For quality control purposes, we filtered out sequences of incorrect length. The number of unique sequences in the randomized 40 nucleotides was computationally counted after trimming the primer-binding regions for downstream analysis.2) at 25 °C. Each of the aptamer candidates were dissolved in PBSM and 30 μl of 100 nM ssDNA samples were injected for kinetic analysis with 2 min dissociation time. To evaluate dose dependent affinity of aptamer to target, two-fold serial dilutions of the aptamer samples were tested (from 3 nM to 50 nM of AS2).PSA and three counter proteins were immobilized on a CM5 sensor chip  to evaluate the binding characteristics of the aptamer candidates. Amine coupling was performed at a flow rate of 5 μl/min in HBS-EP . The CM5 chip surface was activated for 10 min using 0.2 M EDC/0.05 M NHS. After the injection of 10 μg/ml protein in 10 mM sodium acetate (pH 4.5), 5 μl of 1 M ethanolamine (pH 8.0) was used for blocking the remaining activated groups. Binding analysis was performed at a flow rate of 15 μl/min with PBSM  was used as a reference cell to correct the bulk refractive index by subtracting the response signal of the reference cell from the signals of protein immobilized cells. The dissociation constants of the aptamers were calculated by using BIAevaluation software . For the kinetic analysis, the binding curve of the SPR signal was plotted in a Michaelis-Menten equation of  How to cite this article: Park, J.-W. et al. Acousto-microfluidics for screening of ssDNA aptamer. Sci. Rep.6, 27121; doi: 10.1038/srep27121 (2016).


Dear Editor,Hepatitis E virus (HEV), a member of the genus Hepevirus, is a non-enveloped virus with a positive stranded RNA genome that is approximately 7.2 kb in length . It has