However, viral titers decreased precipitously when stored at room temperature; no virus was detectable after one day on all materials tested. A more sensitive immunoplaque assay was able to detect virus from Styrofoam, metal, and plastic at 20 days post application, representing a 3-log loss of input virus on fomite materials. Recovery of infectious PEDV from Tyvek® coverall and rubber was above detection limit at 20 days. Our findings indicate that the type of fomite material and temperatures impact PEDV stability, which is important in understanding the nuances of indirect transmission and epidemiology of PEDV.Indirect transmission of porcine epidemic diarrhea virus (PEDV) ensues when susceptible animals contact PEDV-contaminated fomite materials. Although the survival of PEDV under various pHs and temperatures has been studied, virus stability on different fomite surfaces under varying temperature conditions has not been explored. Hence, we evaluated the survival of PEDV on inanimate objects routinely used on swine farms such as styrofoam, rubber, plastic, coveralls, and other equipment. The titer of infectious PEDV at 4 °C decreased by only 1 to 2 log during the first 5 days, and the virus was recoverable for up to 15 days on Styrofoam, aluminum, Tyvek Coronaviridae. This enveloped RNA virus causes highly contagious viral enteritis in swine [Porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus in the family in swine ,2. The vin swine . From 19in swine ,4,5,6,7.in swine ,9. The vin swine . Over eiin swine ,12,13,14Transmission of PEDV primarily occurs by the fecal-oral route, but indirect transmission can occur when an animal comes in contact with inanimate objects (fomites) contaminated with the feces of PEDV-infected animals. Disease outbreaks primarily occur in early fall and winter, but the rationale for seasonality remains unclear . Previoug for 10 min, the supernatant was aliquoted and stored at −80 °C. Virus titration was performed on Vero 76 cells’ monolayers contained in 96-well microtiter plates using serial 5-fold dilutions of samples. Virus titer was calculated by the Spearman-Kärber method [50)/mL.A strain of PEDV  was propagated on Vero 76 cells  using Dulbecco’s modified Eagle medium (DMEM) supplemented with 0.5 μg/mL TPCK-trypsin  and 0.3% tryptose phosphate broth . The virus was harvested three days post infection (d.p.i.) using one freeze-thaw cycle. After centrifugation at 500× r method  and expr2) of various fomite materials were cut to fit into individual wells of Costar® 24-well cell culture plates . Materials used were Styrofoam, nitrile gloves, cardboard, aluminum foil, Tyvek® coveralls, cloth, metal, rubber, and plastic. Cell culture-grown stock virus was applied on each type of material contained in triplicate wells (200 μL of virus containing 2.1 × 106 TCID50/mL). Virus applied on the bottom of 24-well plate without any fomite material served as control. The applied virus was air dried for 2 h in a biosafety cabinet (BSC). Sets of virus-contaminated fomites were stored at either at room temperature (~25 °C) or at 4 °C. At various times thereafter , the surviving virus was eluted from each material in 1 mL of 3% beef extract −0.05 M glycine buffer placed in 50 mL centrifuge tubes. After thorough mixing for 20 s on a vortexer , the eluate was filtered through a 0.22 μM membrane filters  followed by virus titration on Vero cell monolayers. Virus stability on each fomite material was assessed as three independent replicates for all the time points tested. Separate fomite samples were used at every time point, and eluates were processed independently for quantification of infectious virus and viral RNA. To compare the survival of cell culture-adapted virus with that of virus contained in fecal material, a separate experiment was done. Fecal material obtained from PEDV negative pigs was spiked with 200 μL of cell culture propagated PEDV (2.1 × 106 TCID50/mL). After thorough mixing, contaminated fecal matter was applied in 200 μL amounts on fomite materials in triplicate. Virus was eluted and titrated as described above for cell culture derived virus.Circular pieces . Primer pair was designed to amplify a portion of the PEDV S gene with the following sequences: Forward 1910: ACGTCCCTTTACTTTCAATTCACA and Reverse 2012: TATACTTGGTACACACATCCAGAGTCA. PCR amplification was quantified using a FAM labeled probe 1939: FAM-TGAGTTGATTACTGGCACGCCTAAACCAC-BHQ. The primer and probe were added to AgPath-ID™ One-Step RT-PCR reagents  along with 5 μL of extracted total RNA followed by amplification with 7500 Fast Real-Time PCR System  using the following conditions: reverse transcription at 48 °C for 10 min; denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s; and annealing at 60 °C for 45 s.Immunoplaque assays were performed monolayers of Vero cells contained in 24-well tissue culture plates. Duplicate wells were infected with 200 μL of 10-fold serially diluted sample eluate or stock PEDV (positive control). Plates were incubated for 1 h at 37 °C, and the inoculum was removed and replaced with infection medium with or without 1% agarose. At 24 h post infection (without agarose overlay) or 72 h post infection (with agarose overlay), the medium was removed from the wells and the cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. All subsequent washes and incubations were done in PBS, containing 5% normal goat serum and 0.3% triton X-100. After three washes and blocking for an hour in buffer, plates were incubated overnight at 4 °C with one of the following primary antibodies: (i) mouse monoclonal antibody for PEDV spike protein ; or (ii) mouse monoclonal antibody for PEDV spike protein . Wells were then washed three times with wash buffer and incubated for 1 h at room temperature with alkaline phosphatase conjugated anti-mouse IgG secondary antibody . After three washes in buffer, plates were incubated for approximately 20 min with 1-Step™ NBT/BCIP substrate solution . Immunostained cells were observed under a light microscope .50 assay, immunoplaque assay, and real-time quantitative RT-PCR. An ANOVA was used to determine statistical significance between data sets using GraphPad Prism 7 software .Results are presented as mean ± SEM of the three independent replicates of commonly used fomite materials on a farm. Virus sample eluted from each fomite replicate was processed independently by TCID2 TCID50/mL), while all other materials tested had ≥103 TCID50/mL of residual virus. Infectious PEDV was recovered after 15 days at 4 °C on Styrofoam, aluminum foil, Tyvek® coverall, cloth, and plastic, with titers between 102 to 103 TCID50/mL. However, virus recovery from nitrile gloves, cardboard, metal, and rubber was below the detection limit of the assay (2 × 102 TCID50/mL) after 15 days at 4 °C.Infectious PEDV was recovered from fomite materials for up to 15 days post application at 4 °C; only 1 to 2 logs of virus were inactivated during the first 5 days post application . VaryingOn the other hand, PEDV survival decreased precipitously at room temperature within 1 to 2-days post application, losing 2 to 4 log titers within 24 h .® coverall, metal, rubber, plastic, cardboard, and cloth showed no significant differences between the materials at RT, suggesting that storage temperature had a substantial influence on virus survival.Infectious PEDV was not recovered from any fomite material after 2 days at RT. Virus recovery from surfaces of Styrofoam, nitrile gloves, aluminum foil, TyvekWe used a highly sensitive immunodetection method to identify PEDV positive plaques on Vero cells. The lower limit of detection by this method is 24 focus forming units (FFU)/mL. Positive plaques stain purple after immunostaining with an antibody to PEDV spike protein, followed by NBT/BCIP substrate . This as3 FFU/mL were observed in eluates from Styrofoam, metal, and plastic, representing a 3-log virus inactivation after 20 days. The surviving virus on Tyvek® coverall and rubber surfaces was moderately above detection limit (24 FFU/mL). In contrast, no plaques were detected from fomite materials stored at RT for 48 h post application (data not shown).Using the immunoplaque assay, we re-tested virus stability on certain fomite materials after 20 days of storage at 4 °C. Varying levels of viable PEDV were detected . Titers 50 or immunoplaque assay. In fact, eluates from fecal samples induced remarkable cell toxicity, making the infectious virus assays difficult to interpret. However, viral RNA was detected in these samples after 2 days at RT and 20 days at 4 °C; the RNA levels were similar to those of input virus indicating no significant changes in viral genome levels in feces  at 20 days. The rate of loss in infectivity was uniform on Styrofoam, aluminum, plastic, and Tyvek® coverall, whereas viral infectivity on metal, nitrile gloves, and rubber dropped rapidly after 5 days at 4 °C. Furthermore, at 0 days post application , there was decreased virus recovery from cardboard and cloth, indicating that the composition of the fomite and the porosity of the PEDV-contaminated materials may influence virus survival.The type of fomite material also has implications on viral stability. For example, the survival of PRRSV (porcine reproductive and respiratory syndrome virus) differed in different types of materials including solid, porous, and liquid substances . We foun50 units infects 100% of animals [Our results are consistent with previous studies on virus survival on glass, stainless steel, and plastic for up to 10 days ,27. Spec animals .PEDV transmission appears to be relatively effective via transportation vehicles ,30 and fIn this study, viral RNA copy numbers did not correlate with the cell-based assays. It is possible that PEDV infectivity is destroyed by factors that affect the integrity of the viral envelope without affecting RNA degradation at RT. In a recent study, it was shown that PEDV viral RNA steadily degrades over time at temperatures above 37 °C in cell culture medium, but the rapid decline of viral RNA that is detected by PCR occurs only at temperatures higher than 60 °C . AlthougBesides temperature, the stability of PEDV on fomite is also dependent on presence or absence of other organic material and pH. Although we used both cell culture propagated PEDV and PEDV spiked fecal material on fomites to mimic actual situation in the field, we were unable to determine the survival of the virus in PEDV-spiked fecal material, since the eluates from fomites with feces were not suitable for infectious virus titer due to the cytotoxic effect of the fecal content.In conclusion, our findings provide a new perspective on how fomite material and temperature impact viral stability over time, indicating the significance of understanding the nuances of indirect transmission in the epidemiology of PEDV.


The last case of natural smallpox was recorded in October,1977. It took humanity almost 20 years to achieve that feat after the WorldHealth Organization had approved the global smallpox eradication program.Vaccination against smallpox was abolished, and, during the past 40 years, thehuman population has managed to lose immunity not only to smallpox, but toother zoonotic orthopoxvirus infections as well. As a result, multipleoutbreaks of orthopoxvirus infections in humans in several continents have beenreported over the past decades. The threat of smallpox reemergence as a resultof evolutionary transformations of these zoonotic orthopoxviruses exists.Modern techniques for the diagnostics, prevention, and therapy of smallpox andother orthopoxvirus infections are being developed today. In the 20th centuryalone, over the almost 80 years during which mass vaccination against smallpoxand an intensive anti-epidemic campaign were being conducted, 300 millionpeople still died as a result of the disease[Smallpox .This breakthrough event took place almost a century before the kingdom ofviruses was discovered[In 1796, the English physician Edward Jenner proposed a method for protectionagainst smallpox via the inoculation of the infectious material obtained fromcows with a smallpox-like disease. This method became known asscovered.After its introduction in 1919, mandatory smallpox vaccination in Russia , a vast country with manifold geographic conditionsranging from high-mountain expanses and deserts to northern tundra andoutlandish taiga areas and home to dozens of nationalities differing intraditions, rites, and religious practices, made it possible to eliminatesmallpox morbidity by 1936 [th century. However, smallpoxoutbreaks were still recorded each year in 50–80 countries even in the1950s. In addition, the foci of endemic smallpox in Asia, Africa, and SouthAmerica posed a constant threat of importation to countries already free of thedisease.This dangerous, highly contagious disease was eradicated in many developedcountries in the first half of the 20thWorld Health Assembly (WHA). The corresponding resolution implying completesmallpox eradication was adopted at the 7th WHA plenary meeting on June 12, 1958[Based on an analysis of the tremendous scientific and organizational expertiseon smallpox eradication accumulated in the Soviet Union, V.M. Zhdanov suggestedinitiating a worldwide program of smallpox eradication at the 9This marked the start of an unprecedented international program of globalsmallpox eradication under the aegis of the World Health Organization (WHO).The Soviet Union not only played a key role in initiating the eradicationprogram, but it was also an important backer at all stages of itsimplementation in subsequent years. In 1958, the year of its inception, theSoviet Government offered 25 million doses of dry smallpox vaccines to the WHO,which was then delivered to different countries. In 1960, a laboratory forlarge-scale production of the vaccine, in compliance with the WHO requirements,was organized at the Institute of Viral Preparations . Thislaboratory subsequently became a center where professionals from differentcountries came to master smallpox vaccine manufacturing. A total of over 1.5billion doses of the smallpox vaccine produced in the Soviet Union were usedfor mass vaccination in 45 countries over 20 years of the internationalsmallpox eradication program. This represents one of the key roles played bythe Soviet Union in the global smallpox eradication[The Laboratory for Smallpox Prevention at the Institute of Viral Preparationsplayed an important role in global smallpox eradication and led to theestablishment of the International Reference Center for Smallpox. NumerousSoviet experts were trained at the Center before visiting smallpox-endemiccountries and received the necessary preparation for practical work.rd WHA declared that people throughout the globe had overcomesmallpox. This was the first, and yet only, victory of the world community overa highly dangerous infectious human disease[Thanks to the joint efforts of the world community in anti-epidemic control andmass anti-smallpox vaccination under the Intensified Smallpox EradicationProgramme approved by WHO in 1966, the last natural case of smallpox wasrecorded in Somalia in October, 1977. Based on the statement made by the GlobalCommission for the Certification of Smallpox Eradication on May 8, 1980, the33Once smallpox had been eradicated, the number of laboratories that stored asmallpox virus named variola virus (VARV) was reduced to stave off the risk ofits accidental spread. As early as 1981, only four such laboratories remained and their number was reduced to two in 1984. The latter twolaboratories, namely, the Institute of Viral Preparations  and Centers for Disease Control and Prevention  got the status of WHO Collaborating Centers for Smallpox and OtherPoxvirus Infections [th meeting of the WHO Committee onOrthopoxvirus Infections  to destroy the collections of VARVstrains and their genomic DNAs. Taking into account the planned destruction ofVARV collections, it was necessary to reliably preserve the genetic material ofvarious VARV isolates in biologically safe form, as an issue of paramountimportance for future research. In order to preserve information about thisunique virus, the WHO Advisory Board deemed it necessary to sequence the VARVgenome [Despite strict WHO control, these two repositories of live VARV strains wereregarded as a source of potential biological threat. Correspondingly, adecision was made at the 4National Program for Conservation of Genetic Material ofthe Russian Collection of Variola Virus Strains with L.S.Sandakhchiev, director general of the Scientific and Production AssociationVector (VECTOR), and O.G. Andzhaparidze, the director of IVP, as scientificsupervisors and S.N. Shchelkunov and S.S. Marennikova as principal researchers.Correspondingly, A.I. Kondrusev, a deputy minister of public health in theSoviet Union, and Yu.T. Kalinin, a deputy minister of medical industry,approved the th meeting of the WHO Committee onOrthopoxvirus Infections approved the national programs for research into theVARV genome proposed by Russia  and the United States . In May 1991, the WHO Commission inspectedthe VECTOR’s laboratory headed by S.N. Shchelkunov and officiallyapproved the cloning of VARV DNA fragments and their sequencing.In December 1990, the 5Variola virus. Twosubspecies are commonly distinguished: V. major, causing thedisease with a mortality rate of 5–40% and V. minor,with a lethal outcome of less than 2% [Orthopoxvirus belonging to the familyPoxviridae. This genus also includes the zoonotic species Monkeypoxvirus (MPXV), Cowpox virus (CPXV), Vacciniavirus (VACV), Buffalopoxvirus , and Camelpoxvirus (CMLV), all able to infect humans[The species specific name of VARV is  than 2% .VARV ist humans.major strain isolated inIndia in 1967 during a smallpox outbreak with a mortality rate of 31%, performa computer analysis of the sequencing data[major strain,Bangladesh-1975, isolated during a smallpox outbreak with a mortality rate of18.5% [By mid-1992, Russian scientists were first to successfully complete the genomesequencing of a highly virulent VARV ing data-16, and ing data, 18.Theof18.5% . Subsequof18.5% , 22.th Meeting of the WHO Committee onOrthopoxvirus Infections  that the VARVDNA stocks should be stored in two international repositories, namely, VECTOR(by that time with the status of State Research Center of Virology andBiotechnology) and CDC (United States).It was decided at the 6minor strain,Garcia-1966, was sequenced and analyzed(table)by collaborating teams from VECTOR and the CDC[The complete genome of a low virulent VARV  the CDC.Taking into account the potential threat related to manipulations of live VARVin Moscow, the collection of VARV strains was transferred from the Institute ofViral Preparations to VECTOR  in September, 1994,under a joint order from the Russian Ministry of Health and Medical Industry,the Ministry of Science, the State Committee on Sanitary and EpidemiologySurveillance, and the Academy of Medical Sciences.The WHO officially registered the organization of the WHO Collaborating Centerfor Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and DNA atSRC VB VECTOR in June 1997, after a WHO Commission, in 1995, had inspected thelaboratory facilities providing the highest degree of physical safety intendedfor this purpose. The right of VECTOR to keep the repository of VARV strainsand their genomic DNAs was officially approved by WHA Resolution no. 49.01 andconfirmed by later Resolutions nos. 52.10, 55.15, and 60.1.The WHO Advisory Committee on Variola Virus Research was organized in 1999 tooversee manipulations with VARV and holds annual meetings for all expertsinvolved in relevant studies and in the development of diagnostic, prevention,and therapeutic tools for smallpox and other human orthopoxvirus infections.Table). The analysis of the completeVARV, MPXV, CPXV, VACV, and CMLV genomes made it possible to establish that the CPXV DNAis not only the longest among the studied orthopoxviruses, but also contains all thegenetic elements characteristic of the remaining orthopoxvirus species[To gain insight into the evolutionary interactions of different orthopoxvirusspecies, it became necessary to compare their genomes. The VECTOR’s teamwas the first to sequence the genome DNAs of CPXV and MPXV species, 27-31.  species, 26, 32.The accumulated data laid the basis for the pioneering comparative analysis ofthe genomic strategies utilized by all orthopoxvirus species pathogenic forhumans, the first phylogenetic studies of this virus group, and the discoveryof their evolutionary relationships. However, that data has not yet allowed usto date the molecular evolution of orthopoxviruses and, in particular, VARV, 33-36.The issue of dating the VARV molecular evolution essentially shifted when theVECTOR and CDC teams designed a method allowing one to detect geneticdifferences between VARV strains. The method utilizes complete VARV genomes andincludes long-distance polymerase chain reaction (LPCR) of overlapping genomicsegments of the virus’ DNA (with a length of 10 kbp and longer) withsubsequent hydrolysis of the synthesized amplicons by frequently cuttingrestriction endonucleases, electrophoresis, and a computer-aided analysis ofrestriction fragment length polymorphism (RFLP). This relatively simpleapproach (the LPCR–RFLP assay), which is close to sequencing in itsinformation content , has for the firsttime made it possible to discover detailed differences between the genomes of63 VARV strains from the Russian and U.S. collections isolated in severalgeographic regions and in different years. Phylogenetic analysis of the RFLPdata for viral DNAs allowed us to pioneer the discovery that the West Africanand South American VARV strains form a separate subtype (clade) thatsignificantly differ in their genome organization from the remaining, studiedgeographic variants of VARV . It iseSequencing of the complete genomes of a large set of VARV strains isolated indifferent years and geographic regions ,as wellTaking into account the fact that smallpox vaccination in several cases hadadverse side effects, the WHO recommended ceasing vaccination after 1980 in allcountries. The result of this decision was that mankind lost its collectiveimmunity not only to smallpox, but also to other zoonotic orthopoxvirusinfections. The ever more frequently recorded human cases of zoonoticorthopoxvirus infections force us to revisit the problem of possible smallpoxreemergence resulting from a natural evolution of these viruses, 43.An important feature of VARV is its ability to infect only humans and theabsence of a natural reservoir . One should keep inmind that VARV infection of a human can in many cases (up to 40% and more)result in a lethal outcome-4.MPXV causes a human disease that resembles smallpox in its clinicalmanifestations and also may result in a lethal outcome in up to 10% of cases.The major difference between the human monkeypox and smallpox consists in a lowhuman-to-human transmission efficiency of the former, which so far hasprevented the development of local monkeypox outbreaks into epidemics. HoweverMPXV in the long-term absence of population-scale vaccination and an increasedrate of human monkeypox cases can well acquire the ability to spread from humanto human more efficiently, as is characteristic of VARV. If this happens,mankind will face a much more complex problem as compared to smallpoxeradication. First and foremost, this will have to do with the fact that MPXV,unlike VARV, has a natural reservoir; namely, an abundant African rodentspecies .Other zoonotic orthopoxvirus species typically cause sporadic human infections with a benign outcome in most cases, 9,12. As mentioned above, the comparative analysis of the genomes of VARV and thezoonotic orthopoxviruses pathogenic for humans has shown that CPXV has thelargest genome containing all the genes characteristic of the remainingorthopoxvirus species. Part of the genes in other orthopoxviruses is broken ordeleted, and individual orthopoxviruses have species-specific differences intheir set of retained genes. These data support the concept of reductiveevolution of orthopoxviruses, according to which the loss of genes plays animportant role in the evolutionary adaptation of an ancestral virus to acertain host species, as well as in the emergence of new virus species, 50.VARth century thanks to the jointefforts of medical professionals and scientists from many countries under theaegis of the WHO.An analysis of available archive data on smallpox epidemics, the history ofancient civilizations, and the most recent data on the evolutionaryrelationship between orthopoxviruses has allowed us to hypothesize that VARVcould have repeatedly reemerged via evolutionary changes in a zoonotic ancestorvirus and then disappeared because of an insufficient population size ofisolated ancient civilizations .Only thTherefore, the reemergence of smallpox or a similar human disease in the futurein the course of a natural evolution of currently existing zoonoticorthopoxviruses is not impossible. Correspondingly, it is of utmost importanceto develop and widely adopt state-of-the-art methods for an efficient and rapidspecies-specific diagnosis of all orthopoxvirus species pathogenic for humans,including VARV. It is also important to develop new safe methods for theprevention and therapy of human orthopoxvirus infections.The characteristics of orthopoxvirus infections are similar externalmanifestations, including skin lesions; however, experience has shown thatclinical diagnosis of these diseases is frequently erroneous, 4.The advent of the polymerase chain reaction technique has resulted incutting-edge methods that allow for the detection and identification of traceamounts of microorganisms in assayed samples with a high specificity and over ashort time . MoreoveIn the case of orthopoxviruses pathogenic for humans, test kits that providegenus-specific DNA identification for an assayed virus with concurrentspecies-specific differentiation are a priority. The VECTOR team was the firstto elaborate such methods based on classical multiplex PCR, 53and The method that utilizes oligonucleotide microarrays is also based on PCR; inthe assay, the synthesized DNA amplicons are identified by hybridization withspecific oligonucleotides immobilized on a support in a particular order. TheDNA preparations to be assayed in hybridization are fluorescently labeled.After hybridization and washing, the microarray is analyzed with the help of alaser scanner and the recorded fluorescence data for each cell of themicromatrix are processed using specialized software. Similar to classical PCR,this method can allow one to detect trace amounts of the analyte in a specimen.One of the important advantages of oligonucleotide microarrays is thepossibility to simultaneously analyze a multitude of genetic loci, therebyconsiderably increasing the reliability of the method.Different variants of diagnostic oligonucleotide microarrays have been designedfor species-specific diagnosis of orthopoxviruses-62.The development of next-generation sequencing technologies would makes itpossible to obtain the complete genome nucleotide sequence of a researchsubject in short order. Genome-wide sequencing of isolated viruses in the caseof unusual orthopoxvirus infections is an ever more frequent situation, 64.TheThe first-generation smallpox vaccine was a VACV preparation produced bypropagating the virus on calf  skin. Today, VARV vaccinestrains are produced in mammalian cell cultures and are referred to assecond-generation smallpox vaccines . AlthougThird-generation attenuated smallpox vaccines are produced via multiplepassaging of a VACV strain in the cell culture of a heterologous host. Forexample, the best studied third-generation vaccine, MVA, is produced bymultiple passages of the VACV strain Ankara in a chick fibroblast culture. TheMVA strain genome has accumulated numerous mutations and long deletions thatdistinguish it from the initial VACV strain. MVA is unable to replicate in mostmammalian cells, including human cells .The vaccine based on the VACV strain MVA (Imvanex/Imvamune) has undergonenumerous clinical trials, including studies in subjects with atopic dermatitisand HIV -70. ThisB5R gene that encodes a proteinessential for extracellular enveloped virion formation [Another third-generation smallpox vaccine, LC16m8, licensed in Japan, wasproduced from VACV strain Lister via multiple passages in a primary rabbitkidney cell culture at a decreased temperature (30°C). Clinical studieshave demonstrated a considerable reduction in the number of adverse sideeffects compared to the conventional Lister-based vaccine. The resultingattenuation of this vaccine strain is mainly due to a mutation (singlenucleotide deletion) in the ormation , 75. Theormation , 77.A new approach to the production of fourth-generation attenuated smallpoxvaccines consists in genetic engineering of variants with impaired genes thatcontrol the host’s protective response to virus infection, the range ofsensitive hosts, etc. by introducing targeted deletions/insertions. The beststudied variant of such VACV is strain NYVAC, with a deleted block of 12 genesand six additional individual damaged genes. The NYVAC strain inducesconsiderably weaker immunity in humans as compared to the classical Lister orDryvax vaccine, including the inability to induce A27-specific antibodies,which are necessary for efficient neutralization of a VACV infectious form, theintracellular mature virus , 79.A35R gene yielded another highlyimmunogenic attenuated strain, VACdelta6 [In Russia, a highly attenuated VACV variant was produced by successiveintroduction of targeted deletions/insertions into five individual genes ofstrain LIVP . AdditioACdelta6 ,which iACdelta6 .in vitro antiviral activity using animalmodels, first and foremost, intranasal or aerosol infection of mice with CPXVand monkeys with MPXV [Chemotherapeutics are no less important in the treatment of human orthopoxvirusinfections, and the search for such drugs over the past 20 years has been asuccess. Since there are no adequate animal models for smallpox, potentialanti-smallpox drugs are tested in surrogate smallpox animal models . The inhith MPXV , 85. Rabith MPXV -88. HoweCidofovir, an antiviral nucleotide analog (brand name Vistide) officiallyapproved for clinical use against cytomegalovirus retinitis and acting as aninhibitor of virus DNA polymerase, was the first compound intensively studiedas an anti-orthopoxvirus drug .CidofovST-246, a compound that blocks the final stage in the assembly of intracellularenveloped virions and prevents the release of the virus from an infected cell, 90, is The search for new anti-orthopoxvirus chemotherapeutic agents with othermolecular targets is in progress ,94.The analysis of the genome organization of orthopoxviruses pathogenic forhumans and their patterns of evolution suggest the fundamental possibility thatsmallpox or a similar human disease can emerge in the future via a naturalevolution of extant zoonotic orthopoxviruses. Cessation of anti-smallpoxvaccination and the resulting loss of collective population immunity not onlyto smallpox, but also to other orthopoxvirus infections creates conditions thatpromote the spread of zoonotic orthopoxviruses among people, therebypotentially enhancing the selection of virus variants highly pathogenic forhumans and epidemically dangerous. However, the situation today does not lookirredeemable and radically differs from the events far past, when man had nocontrol over infections. Today, most outbreaks of orthopoxvirus infections indomestic animals and humans are registered and investigated; in addition, theefficient international system for clinical sampling and identification ofinfectious agents has been validated and anti-epidemic activities and protocolsfor mass vaccination were developed during the implementation of the globalsmallpox eradication program .The recent efforts at the WHO are directed towards developing state-of-the-artmethods for rapid VARV identification and designing next-generation safeanti-smallpox vaccines and chemotherapeutic agents against VARV and otherorthopoxviruses .The studied vaccines and chemotherapeutics are not strictly species-specificwith respect to orthopoxviruses pathogenic for humans and, thus, are applicableto outbreaks caused by any orthopoxvirus species. Taking into account the abovesuccinct information, it results that diagnostic methods should be focused onrapid identification not only of VARV, but also of MPXV, CPXV, VACV, and CMLV. The recde novosynthesize the complete horsepox virus genome and obtain a live virus[Phenomenal advance in synthetic biology has made it possible to ve virus. This su


Information about the Zika virus disease incubation period can help identify risk periods and local virus transmission. In 2015–2016, data from 197 symptomatic travelers with recent Zika virus infection indicated an estimated incubation period of 3–14 days. For symptomatic persons with symptoms >2 weeks after travel, transmission might be not travel associated. Aedes spp.mosquitoes. Transmission can also occur through occupational laboratory exposure and by intrauterine, intrapartum, or sexual routes   by using the dic.fit function in the coarseDataTools package, which uses methods detailed by Reich et al. . We then performed a secondary analysis of persons with confirmed Zika virus infection and <2 weeks of travel (secondary case set), enabling evaluation of our estimates by using more stringent case definition requirements. A confirmed case of Zika virus disease was illness in a symptomatic person with a sample that was either Zika virus RNA positive or Zika or dengue virus IgM positive with neutralizing antibodies against Zika virus only.From January 1, 2015, through June 23, 2016, we identified 337 persons with evidence of recent Zika virus infection. Of these, we excluded 140 (42%) because they did not meet the study criteria . Among tThe Weibull distribution fit our data best (parameter estimates in Of the 112 (57%) persons who had traveled for <2 weeks, cases were confirmed for 79 (71%). The age and sex distributions for these patients did not differ significantly from those of the primary case set   days  Table 2 –On the basis of our analysis, we estimate that the incubation period for Zika virus is 3–14 days. We expect symptoms to develop within 1 week of infection for 50% and within 2 weeks for 99%. Our estimates for Zika virus incubation period are similar to those reported for other flaviviruses  or local vectorborne transmission.Parameter and incubation period quantile estimates for persons with confirmed Zika virus disease and for all persons with Zika virus disease, United States, January 1, 2015, through June 23, 2016.


Various viruses leave their sequences in the host genomes during infection. Such events occur mainly in retrovirus infection but also sometimes in DNA and non-retroviral RNA virus infections. If viral sequences are integrated into the genomes of germ line cells, the sequences can become inherited as endogenous viral elements (EVEs). The integration events of viral sequences may have oncogenic potential. Because proviral integrations of some retroviruses and/or reactivation of endogenous retroviruses are closely linked to cancers, viral insertions related to non-retroviral viruses also possibly contribute to cancer development. This article focuses on genomic viral sequences derived from two non-retroviral viruses, whose endogenization is already reported, and discusses their possible contributions to cancer. Viral insertions of hepatitis B virus play roles in the development of hepatocellular carcinoma. Endogenous bornavirus-like elements, the only non-retroviral RNA virus-related EVEs found in the human genome, may also be involved in cancer formation. In addition, the possible contribution of the interactions between viruses and retrotransposons, which seem to be a major driving force for generating EVEs related to non-retroviral RNA viruses, to cancers will be discussed. Future studies regarding the possible links described here may open a new avenue for the development of novel therapeutics for tumor virus-related cancers and/or provide novel insights into EVE functions. Viruses can deposit their sequences into the host genome during infection. Consistently, animal genomes contain many viral-related sequences, called endogenous viral elements (EVEs) . EVEs arFigure 1A). First, the inserted sequences in the vicinity of an oncogene may function as a promoter for the oncogene. Second, such events may inactivate tumor suppressor genes via insertional mutagenesis. Third, such integrated sequences may induce genomic instability via homologous recombination  . HBV is e family , while He family . Both vie family . Consiste family . The rabe family . Althouge family . HCV cDNe family , furtherFigure 1B). One of the first cases is the recurrent insertion into the telomerase reverse transcriptase (TERT) gene  gene . TERT exRT) gene . HBV inselopment . The secelopment .HBx-L1, is an example of the third possible mechanism described above  in the human genome, hsEBLN-2 is most closely linked to cancer. Whole exome sequencing using two sibling pairs of non-smokers with lung adenocarcinoma reveals that a truncated mutation in hsEBLN-2 is only detected in affected siblings (4LKLY8) at the N-terminal. Furthermore, the hsEBLN-2 protein was shown to be expressed and to interact with several other host proteins .Endogenous bornavirus-like elements are the only non-retroviral RNA virus-derived EVEs found in the human genome, although DNA virus-derived EVEs are also found in the human genome . The majsiblings . The autsiblings . The lossiblings . hsEBLN-siblings . Gene onproteins . Becauseproteins , hsEBLN-Figure 1C). Both studies demonstrated that hsEBLN-1 silencing increases the proportion of cells in the G2/M phase. hsEBLN-1 knockdown cells exhibit microtubule and centrosomal splitting defects  that encodes 366 amino acids, which is comparable with the full-length BDV N protein . Despite defects . Proteom defects . Among t defects . In addio glioma . This obCOMMD3 (COMMD3 gene was downregulated. The effect of hsEBLN-1 RNA expression on the COMMD3 locus was abrogated by treatment with siRNA against hsEBLN-1 RNA. These results suggest that hsEBLN-1 RNA may function as a lncRNA that scaffolds transcriptional and/or epigenetic repressors for the COMMD3 gene and suppress its expression. Although we cannot exclude the possibility that hsEBLN-1 functions as a cis-regulatory DNA element or a protein acting on this locus in trans, our data using siRNA and cytoplasmic localization of the hsEBLN-1 protein strongly suggest a role for hsEBLN-1 as a lncRNA (COMMD3 gene encodes a protein that can interact with and inhibit the NF-kB pathway (COMMD3 gene was reported in a particular type of leukemia (COMMD3 gene and thereby potentiate the NF-kB pathway (We have previously demonstrated that hsEBLN-1 can modulate the expression of its neighboring gene, COMMD3 . When tra lncRNA . The COM pathway , which rleukemia . EBLN in pathway . Cancer  pathway . Taken tAs described above, non-retroviral RNA virus-related sequences in the genome are possibly generated by a retrotransposon machinery . In otheThis article has presented a current view of the possible contributions of hepadnavirus and bornavirus insertions in the genome to cancer formation. The presented lines of evidence suggest potential links between these viral sequences and cancers. However, current knowledge in this field is still poor, and there are many questions to be addressed. Although several genes recurrently targeted by HBV insertions have been identified, the precise role of most of them in tumorigenesis remains unclear. Among the HBV integration sties identified so far, only a limited number of cellular-HBV chimeric proteins/transcripts have demonstrated the oncogenic potential. Further accumulation of examples of recurrent HBV insertion sites in the host genome or recurrent chimeric transcripts specific to hepatitis virus-related HCC will be promising to understand the contribution of HBV insertions to HCC etiology. Regarding links between EBLs and cancers, the information is more limited. Epidemiological studies on the links between EBL mutations and cancers are clearly required. Furthermore, the causal relationship between such EBL mutations and cancers should be demonstrated in future.Although a definitive role for tumor viruses in retrotransposon activation has not been established thus far, investigating a possible link between L1 activation and tumor viruses, especially HBV, would be of considerable interest because L1 hypomethylation or some L1 chimeric transcripts are associated with poor prognosis in HCC . HypometFuture studies regarding the above links may open a new avenue for the development of novel therapeutics, such as epigenetic modification of viral sequences in the genome, for tumor virus-related cancers. Also, such studies will provide novel insights into the biological roles of EVEs in the cells.TH wrote the manuscript, confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID Similar to the manufacturing of antibodies, growth factors or hormones, the production of viral vaccines and vectors for gene therapy in batch cultivation is still the standard technology. Moving from batch to continuous operation can significantly increase productivity, improve the quality of products, and reduce the footprint of equipment and facilities required for production . The upsOne potential platform for continuous production of viruses is a two-stage stirred tank bioreactor (TSB) system. A TSB system consists of one vessel for continuous propagation of cells connected in series with a second vessel, where the desired product is obtained , 4. The Babesia bovis [One virus that has a great potential for expression of recombinant antigens or as a viral vaccine vector is Modified Vaccinia Ankara (MVA) virus, a DNA virus that can accommodate large recombinant inserts and is described to be safe for humans and animals . Recombiia bovis , have beia bovis . Recentlia bovis , which mWhen a fraction of a culture is replaced with fresh media at regular time intervals, the mode of operation is defined as “semi-continuous”. Compared to fully continuous operation in bioreactors, a scale-down of semi-continuous cultures can be easily achieved for studying the impact of process parameters on cell growth and virus propagation. In addition, semi-continuous cultures can be set to medium exchange regimes that can approach continuous cultivations . To nameHere, we describe results for the establishment of semi-continuous and continuous processes for production of MVA virus , MVA-CR19.GFP) in the avian suspension cell line AGE1.CR.pIX in a chemically defined medium  –30. Firs®R3IGF-I . Cells were inoculated at a concentration of 0.8×106 cells/mL and passaged in shaker flasks  at 37°C, 5% CO2 in air, and 185 rpm [8 virions/mL, TCID50) and MVA-CR19.GFP  with a green-fluorescent-protein insertion cassette were used . Before infection, the virus seed was sonicated in a water bath at room temperature for 1 min. An moi of 0.05 (ratio of the number of infectious virions to the number of cells) was used in all cultivations.The avian cell line AGE1.CR.pIX  was grown in a chemically defined medium CD-U3  supplemented with 2 mM of L-glutamine , 2 mM L-alanine  and 10 ng/mL Long 185 rpm . Previou 185 rpm ,31. The 6 cells/mL [AGE1.CR.pIX cells were cultured in 50 mL working volume (wv) shaker flasks , for batch cultivations. With cell concentrations reaching about 5×10cells/mL , a 1:1 d2 in air. Cells were grown in batch mode for up to 96 h and infection was carried out at an moi of 0.05. Samples of 3–5 mL for measuring cell concentration, viability, pH, metabolite concentrations, and virus concentration in the small cell bioreactor (SCB) and the small virus bioreactor (SVB) were taken twice a day for process monitoring. Small volume losses after sampling were corrected by adding fresh medium. After sampling, medium exchanges and addition of fresh medium were carried out. The volumes exchanged were determined with the following equations:n is the time at sampling “n”, tn-1 the time at sampling “n-1”, DSCB is the dilution rate of SCB, and FM is fresh medium. The dilution rate of SCB (DSCB) was the same in all SSC experiments. The equations were obtained by performing a material balance, where the dilution rate of the TSB system F4/(VCB + VVB) is equal to the dilution rate of CB  compared to batch cultivations  only one cultivation was carried out per SSC experiment (unless the opposite is indicated).A SSC system using two shaker flasks  in series was established as a scale-down model for design and parameter studies regarding TSB optimization. All related abbreviations, volumes and the operation mode of the system are described in f CB see . Based o3, 3 to zero (50 by fluorescence microscopy (described in section 2.4.2). This ratio was determined with the following equation:To evaluate the influence of RT on MVA-CR19 virus yields, the volume of SVB was modified to obtain a RT of 25, 35, and 64 h in the SVB . Also, the effect of omitting the addition of fresh medium to the VB F3,  was eval to zero . The staWith IVP and PEIVP the mean of a technical triplicate. The second criterion used was a genetic analysis of the GFP insertion cassette using a polymerase chain reaction (PCR) protocol (described in detail in section 2.4.2). The samples analyzed with both criteria were taken from the first and last harvest of two SSC cultivations operated over 16 d, one with 25 h RT in the SVB and a second with 40 h RT in the SVB.6 cells/mL and operated at 37°C, 120 rpm with Rushton impellers, 40% oxygen saturation and 850 mL working volume (wv). Oxygen saturation was controlled using pulsed aeration with pure oxygen. The pH value was not controlled and only monitored during the batch phase to avoid values below 6.9. When the cell concentration in the CB reached at least 5.0×106 cells/mL, 350 mL from the CB were transferred to the second vessel VB. Working volumes were corrected to 850 mL in CB and 440 mL in VB by adding fresh CD-U3 medium and continuous culture was initiated 2 h later and maintained without infection for 8 d. The dilution rate of CB (DCB) was as reported previously [VB) was 0.04 h-1 (25 h RT). Temperature of VB was 37°C, oxygen concentration was at 40–50% saturation, and the 440 mL wv was maintained with a dip tube. Before infection, a 1:1 dilution of VB was carried out, and the cultivation continued at 440 mL wv. MVA-CR19 virus was added to VB at an moi of 0.05. The peristaltic pumps used were Ismatec Reglo-Digital MS2/8-160  was established  was used for determining AGE1.CR.pIX cell concentration and viability, with a standard deviation of 5% [A ViCellon of 5% . Glucose50 assay as described previously [50 titer was obtained by measuring cell fluorescence using a fluorescence microscope . The GFP-assay was carried out immediately before performing the regular TCID50 staining procedure. The PCR was carried out by mixing the infected cell suspension (80 μL) with 20 μL of QuickExtract DNA Extraction Solution 1.0  and heated to 65°C for 10 min and to 98°C for 5 min. Of this preparation, 4 μL were used in a PCR reaction in a final volume of 20 μL with 0.15 μL Taq polymerase , 200 nM of each primer, and 125 μM of each nucleotide. The sequence of the primer pairs that span deletion sites II, III and IV of the viral genome have been published previously [The concentration of infectious virus particles was quantified by a TCIDeviously . In addieviously . The expn is the total operational time , 50 of a harvest at time tn , and n. Eqs The productivity of batch, semi-continuous and continuous cultivation systems was determined based on two parameters: time yield (TY) and space-time yield (STY). The following equations were used for all three cultivation modes:×VH,tn)tnSTYtn=∑tVH,tn)×tnWhere tn6 cells/mL and grew in batch mode for 3–4 d until 5.0×106 cells/mL were reached. For the MVA-CR19 virus strain used for infection, TCID50 titers of 3.0×108, 1.0×108 and 0.3×108 virions/mL were obtained at 72 h post infection, as summarized in 50 titer of 1.4×108 virions/mL at time of harvest , the productivity of the hypothetical batch process (see productivity indicators section) was determined as 8.9×108 virions/h (TYB) and 3.4×108 virions/(L h) (STYB) for 17 d of operation (2 batch cycles). For 26 d of operation (3 batch cycles) this corresponds to 8.7×108 virions/h and 2.2×108 virions/(L h) for TY and STY, respectively , these oscillations are most likely due to variations in the cell concentration in the SVB and measurement errors of virus titrations.SVB was infected with MVA virus after 3–4 d of batch growth, and the semi-continuous mode with harvesting was started 12 h p.i. The cell concentration initially also fluctuated in a similar range as for the SCB, but then fluctuations decreased as a consequence of progressing virus replication. Cell viabilities of 90% or more were observed in SVB for the first days, but dropped after 12 d . The virhed data , 31. In hed data , only a 5 virions/mL. The TCID50 titers showed the typical pattern of virus replication, an initial increase followed by a stationary phase. Depending on the RT in the SVB, the increase in virus titers differed significantly. With the lowest RT , the increase was fast and high titers were achieved within 2 d  which showed that addition of fresh medium at time of infection is usually required for obtaining high virus yields in batch processes [50 titers in the stationary phase with fluctuations of less than 2 log units. Taking additionally into account the results of the SSC cultivations, a RT of 25 h was therefore used to scale up the virus bioreactor in the continuous cultivation.Also, to test whether high virus yields can be obtained by reducing the amount of fresh medium added to SVB, one experiment was performed without the addition of fresh medium into the virus vessel SM35-C; , Fig 3. rocesses . Despite9, 4.1×108, and 2.0×108 virions/h for the experiments SM25-B, SM-35-B and SM64, respectively  for experiments SM25-B, SM-35-B and SM64, respectively. These values were in the same order of magnitude as the batch process operated 8 d (5.2×108 virions/(L h), 1 cycle) and 16 d (2.5×108 virions/(L h), 2 batch cycles), respectively. Accordingly, the productivity for SSC systems is similar or better than for batch processes. It has to be taken into account, however, that a batch process is more efficient than SSC for low volume MVA vaccine production (single batch campaigns).As a general tendency, a higher productivity was obtained for experiments with lower RT in SVB. The TY was 1.0×10ectively . The bes50 as well as the GFP-derived TCID50 were determined , the MVA-CR19.GFP virus was propagated for 16 d in SSC, and the TCID25 h RT, . For botectively . At 40 hectively . ConsideIn addition, the genetic stability of MVA-CR19.GFP virus was analyzed using PCR of the first and last harvest of both SSC cultivations. For the two RT experiments, the GFP insertion cassette amplified by this method was visible in the deletion segment 3 (Del 3) of the first (0 d p.i.) and last harvest (16 d p.i.) with a size of 1428 kbp . The fac6 cells/mL in batch operation with cell viabilities well above 90% . Immediately before the continuous culture was initiated, a concentration of about 2.5×106 cells/mL was reached in both vessels by 1:1 dilution with fresh medium. Once the continuous operation was started, the cell concentration in the CB increased initially to 5.0×106 cells/mL, and finally decreased to 3.0×106 cells/mL with 85% viability. Nevertheless, cells could be maintained stable at these viabilities for the rest of the experiment. Also, the continuous process was maintained without infection of VB for the next 8 d . This confirms that SSC could serve as a scale-down model of the TSB system, and is in line with previous work performed by Van Lier et al. who have demonstrated for baculovirus A. californica that semi-continuous cultivations can approximate continuous two-stage cultures [Interestingly, the TSB results resembles some of the results obtained with the semi-continuous cultivation system. Experiments SM25-A and SM25-B were performed with a RT in SVB identical to the VB of the TSB system. Virus titers from SM25-A  showed acultures . In addicultures . Overall9 virions/h, and the STY was 1.7×108 virions/(L h) for a total of 21.7 d of operation (17.7 d of virus propagation). The time course of TY and STY over the production time is shown in 7 virions/mL and 108 virions/mL at toh) are also included. This figure shows that both the TY and the STY of the TSB started below the productivity of a batch. After 7 days, the productivity of the TSB approached the batch process and remained constant. In other words, the TSB system can be equally or more efficient than a batch production system, if more than two batch-cycles are performed  system, was used to support the establishment of a two-stage continuous stirred tank bioreactor (TSB) system that showed stable production of cells over 2–3 weeks of cultivation.A higher residence time in the SSC system resulted in a higher delay before virus titers increased, but had little impact on the final titers of the MVA-CR19 isolate in the stationary phase. A critical parameter for high yields appears to be the incorporation of a stream of fresh medium in the virus bioreactor, to remove spent medium including compounds inhibiting virus replication and to avoid limitation of cell-relevant metabolites during virus replication.Evaluation of continuous production of MVA was facilitated with the SSC system because high virus titers were obtained throughout the process interval without oscillations of infectious units at different residence times. The stable titers suggest that a “von Magnus effect” due to formation of DIPs is highly unlikely for MVA. This observation is also consistent with the results from assays for maintenance of recombinant inserts by titration against the fluorescence GFP transgene and by PCR analysis. These assays demonstrated that MVA is stable for at least 16 d of continuous production.50 titers, again with only small random fluctuations over 18 d of virus production and with productivity indicators comparable to repeated-batch cultivations. However, titers appeared to be lower for the recombinant virus that expresses GFP if the RT was only 25 h. This observation may indicate that MVA-CR19.GFP may have longer replication cycles under the chosen conditions and that the RT has to be adjusted accordingly for scale-up.A scale up to a TSB system for MVA-CR19 virus propagation was successful and showed high TCIDOverall, our results demonstrate that at least some recombinant MVA virus strains can be stably and efficiently propagated in TSB systems designed for continuous and semi-continuous production of MVA-based recombinant vaccines and viral vectors.


Mycoplasma cynos. The isolate, Canis familiaris polyomavirus 1 (DogPyV-1), is a beta polyomavirus whose closest known LT antigen relatives are primate polyomaviruses. We report here the first canine polyomavirus genome, identified by metagenomics in respiratory secretions of two dogs with severe pneumonia, which tested negative for all canine respiratory pathogens except  Mycoplasma cynos on a real-time PCR panel . Additional pathogens tested by PCR included canine circovirus, Leptospira spp., Listeria monocytogenes, Pneumocystis carinii, and P. murina. Microbiological cultures on the secretions remained without aerobic growth. Three dogs from the same household in the New York City area developed respiratory symptoms in November 2015 following the introduction of a Labrador retriever puppy from a pet store. The puppy fell ill with respiratory signs shortly after joining the household but recovered uneventfully. Two unrelated 10-year-old Dachshunds in the household became sick shortly afterward. One Dachshund died, while the second was diagnosed with pneumonia based on thoracic radiography showing severe diffuse pulmonary infiltrates. Despite intensive antibiotic therapy, this dog was later euthanized due to progressive disease. Lung tissues collected from this dog revealed marked neutrophilic bronchointerstitial pneumonia. Respiratory swab samples from both adult Dachshunds were negative for all respiratory pathogens with the exception of Canis familiaris polyomavirus 1 (DogPyV-1) with GenBank accession number KY341899. Retesting of two swabs from the two deceased Dachshunds using a DogPyV-1-specific RT-PCR test confirmed the virus in both respiratory secretions. The presence of DogPyV-1 nucleic acid in lung tissue samples was confirmed using in situ hybridization.Enrichment for viral particle-associated nucleic acids in a respiratory swab from the second Dachshund was followed by unbiased nucleic acid amplification using random RT-PCR and use of an Illumina Nextera kit and sequenced using the MiSeq PE300 platform. Similarity sequence searches against all viral protein sequences in GenBank using BLASTx showed the presence of polyomavirus sequences that could be assembled into a typical 5,136-nucleotide-long circular genome for Polyomaviridae family was revised to accommodate a rapidly growing number of newly discovered viruses in diverse animal species (Betapolyomavirus (Macaca mulatta polyomavirus 1 (Cercopithecus erythrotis polyomavirus 1 (In 2016, the taxonomy of the avirus 1 , Cercopiavirus 1 , Simian avirus 1 , Vervet avirus 1 , Anubis The presence of a novel polyomavirus in both tested Dachshunds from the same outbreak raises the possibility that this virus may be a previously unrecognized canine respiratory pathogen. Further studies are under way to determine the virus’s prevalence, cellular tropism, and association with canine respiratory disease.KY341899.The full-length viral genomic sequence of DogPyV1 was deposited in GenBank under the accession number


Incidence of neurologic manifestations associated with Zika virus infection has been increasing. In 2016, neuropsychological and cognitive changes developed in an adolescent after travel to a Zika virus–endemic area. Single-photon emission computed tomography and neuropsychological testing raised the possibility that Zika virus infection may lead to neuropsychiatric and cognitive symptoms. Although the full clinical spectrum of complications associated with Zika virus disease remains unclear, evidence of an association between Zika virus infection and diseases of the nervous system is growing  results positive for Zika virus. Serologic results were positive for Epstein-Barr virus, consistent with the patient’s history of infection 15 months earlier  Table 1.Symptoms failed to improve, and 13 days after symptom onset, the patient was hospitalized for further work-up. Fourteen days after symptom onset, magnetic resonance imaging of the brain with contrast showed no abnormalities. Serum was positive for dengue virus IgG and IgM  Table 2.After discharge, the patient’s symptoms of regression, disinhibition, and cognitive impairment persisted. Seven weeks after symptom onset, single-photon emission computed tomography revealed mildly heterogeneous cerebral cortical perfusion, with focal moderate-to-severe hypoperfusion in the inferior left frontal region  Figure 2Although we cannot entirely rule out Epstein-Barr virus as a possible trigger, the length of time between previous infection and onset of neuropsychological symptoms would be unusual. In addition, although it is impossible to exclude contributions of coinfection from other mosquitoborne viruses , given the Zika virus positivity on RT-PCR, the patient’s condition met criteria for definitive Zika virus infection and the CSF IgM titer was consistent with CNS involvement of Zika virus. The changes on single-photon emission computed tomographs and neuropsychological test scores raise the possibility that Zika virus infection may trigger neuropsychiatric and cognitive symptoms. Although we cannot prove that the patient’s symptoms were related to Zika virus, clinicians should be aware of this potential association and the value of closely monitoring patients with Zika virus infection.Outpatient and inpatient laboratory results, neuropsychological test results, photograph of rash, and single-photon emission computed tomography and magnetic resonance images for an adolescent returning from a Zika virus–endemic area, 2016.


Aedes aegypti mosquito–human cycle, a logical question, as suggested by in vitro data, is whether dengue virus infections result in antibody-dependent enhancement of Zika virus infections. This review emphasizes the critical role for epidemiologic studies (retrospective and prospective) in combination with the studies to identify specific sites of Zika virus infection in humans that are needed to establish antibody-dependent enhancement as a possibility or a reality. The sudden appearance of overt human Zika virus infections that cross the placenta to damage fetal tissues, target sexual organs, and are followed in some instances by Guillain-Barré syndrome raises questions regarding whether these outcomes are caused by genetic mutations or if prior infection by other flaviviruses affects disease outcome. Because dengue and Zika viruses co-circulate in the urban  Recently, polyclonal and monoclonal dengue virus (DENV)–elicited antibodies have been shown to neutralize or enhance Zika virus infection in vitro , fueled by the ADE phenomenon, occur specifically in DENV-immune persons? If Zika virus infections are enhanced by DENV antibodies, might ADE also occur after administering a dengue vaccine? Indeed, if Zika virus infections can be enhanced by DENV antibodies, might that also be an outcome following administration of a poorly protective Zika virus vaccine? Correspondingly, might Zika virus antibodies enhance DENV infections? These crucial questions require biologically valid answers. What information do we need to answer them, and where shall we start looking?Flavivirus genus, is maintained in complex African zoonotic cycles, spilling from time to time into the Aedes aegypti mosquito urban transmission cycle  epidemiologic evidence demonstrating that a unique syndrome or severe disease is significantly associated with persons who circulate antibodies (presumably “enhancing”) before infection, and 2) evidence of in situ replication by the causative organism in myeloid cells that serve as major targets of cellular infection. DENV disease enhancement was established by recording a strong association between severe disease in humans and a secondary-type DENV antibody response, by direct association of severe cases with sequential DENV infections in prospective cohort studies, and by the occurrence of severe disease during first DENV infections in infants born to dengue-immune mothers  or the appearance of a generalized body rash (humans) (Should Zika virus infect circulating monocytes, this still leaves unanswered the question whether tissue macrophages are an important component of so-called “normal” Zika virus human infections. In A129 mice lacking type 1 interferon receptors, peripheral inoculation of Zika virus produced observable illness and high titers of virus in spleen, liver, and brain, but no attempt was made to identify target cells (


In particular, UPO216-specific antibodies showing no cross-reaction with other serotypes of APMV were detected in four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). These results indicate that the UPO216 virus has antigenically and genetically unique characteristics distinct from known serotypes of APMV and likely has been circulating widely in wild duck species of the Order Anseriformes. Thus, we propose the UPO216 isolate as a prototype strain of a novel APMV serotype (putative APMV-15).In January 2014, a viral hemagglutinating agent named UPO216 was isolated from fecal droppings of wild birds at the UPO wetland in South Korea during an avian influenza surveillance program. Electron microscopy identified the UPO216 virus as an avian paramyxovirus (APMV). Pathogenicity tests and molecular pathotyping revealed that the virus was avirulent in chickens. The UPO216 virus was assigned to a serological group antigenically distinct from known serotypes of APMV  by hemagglutination inhibition test, despite showing weak cross-reactivity with APMV-1 and APMV-9. The UPO216 virus RNA genome is 15,180 nucleotides (nts) in length, encodes 3′-N-P(V/W)-M-F-HN-L-5′ in that order, and shows unique genetic characteristics in terms of genomic composition and evolutionary divergence (0.43 or greater from known serotypes of APMV). Phylogenetic analysis revealed that the UPO216 occupies a branch separate from APMV-1, -9, -12, and -13. Serologic surveillance of wild birds ( Abulavirus within the family Paramyxoviridae. The genomes of APMVs contain a non-segmented negative sense single-stranded RNA ranging from 15 to 17 kb in length and encoding at least six proteins, nucleocapsid (N), phospho- (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and a large polymerase (L). Two additional proteins, V and W, may be produced by an RNA editing event during transcription of the P gene  belong to the genus APMVs are divided into 14 different serotypes (APMV-1 to −14) according to cross-hemagglutination inhibition (HI) testing  in the Falkland Islands in 2007  in France in 2010  in Italy in 2005  have been isolated from wild bird populations. APMV serotypes −10, −11, and −12 were isolated from Rockhopper penguins (The emergence and worldwide spread of the H5N1 highly pathogenic avian influenza virus (AIV) in poultry and wild birds in the early 2000s spurred the development of AI surveillance programs to better understand the ecology of AIVs in wild birds, especially waterfowl, worldwide  in the southern region of South Korea in January 2014. Here, we examined the biological properties and genomic features of this isolate to determine whether the virus is a novel APMV.50), as calculated by the Reed and Muench method  in the Gyeongnam province of Korea during an AI surveillance program in 2014 by inoculation into the allantoic cavities of 9–11-day-old specific pathogen-free (SPF) embryonated chicken eggs  as previously described  continuous sucrose gradient centrifugation at 90,000 × g for 1.5 h at 4°C in a Beckman SW41 rotor . Each fraction was collected and tested for HA activity. HA-positive fractions were diluted in phosphate buffered saline  and precipitated by centrifugation at 26,000 × g for 6 h at 4°C. The pellet was resuspended in PBS and kept at 4°C until use. Purified virus was adsorbed to 300 mesh copper grids at room temperature. The grids were negatively stained with 2% uranyl acetate and examined under a Hitachi 7100 electron microscope  at a magnification of × 100,000.9.0 EID50 per dose) as previously described 1/2, where r1 is the ratio of the heterologous HI titer to the homologous HI titer for virus 1 and r2 is the ratio of the heterologous HI titer to the homologous HI titer for virus 2.To serotype APMVs, a cross-HI test was performed using a reference panel comprising antigens and chicken anti-sera against representatives of APMV-1 to APMV-9  using the method outlined in OIE  terrestrial manuals OIE, . ChickenThe virulence of the virus was determined by the intracerebral pathogenicity index (ICPI) in day-old SPF chicks and the mean death time (MDT) in 9–11-day-old embryonated SPF ECEs, as previously described  according to the manufacturer′s instructions. Complementary DNA synthesis was performed using a LaboPass™ cDNA synthesis kit . A combination of F gene-specific primers and primer walking was used to generate PCR amplicons covering the genome (except for both ends). The sequences of the 3′ and 5′ ends of the genome were amplified using the rapid amplification of cDNA ends (RACE) method  were used for phylogenetic comparison. Editing and sequence analyses were performed using the BioEdit sequence alignment editor Hall, . AlignmeJF950510, AJ88027, and JF893453; APMV-2, accession nos. HM159993, HM159995, and HQ896023; APMV-3, accession nos. EU403085 and EU782025; APMV-4, accession nos. FJ177514 and EU877976; APMV-5, accession no. GU206351; APMV-6, accession nos. JN571486 and GQ406232; APMV-7, accession no. FJ231524; APMV-8, accession no. FJ619036; APMV-9, accession no. EU910942; APMV-10, accession no. HM159995; APMV-11, accession no. JQ886184; APMV-12, accession no. KC333050; APMV-13, accession nos. LC041132 and KX119151; and APMV-14, accession no. KX258200.Genome sequences of representative APMV serotypes were retrieved from GenBank public databases and used for the alignments. The GenBank accession numbers are as follows: APMV-1, accession nos. Anseriformes, Charadriiformes, Ciconiiformes, Columbiformes, and Passeriformes covering 15 species. Sera were heat-inactivated at 56°C for 30 min before use in the serologic assay. Serological HI tests employing four HA units of test antigen in V-bottom microtiter plates were performed as described previously  were considered positive. All tests were repeated twice.A total of 880 serum samples from wild birds in Korea were examined. The serum samples were originally taken from wild birds captured for an AI surveillance program; samples were kept at the AI laboratory, QIA, Korea. The serum samples were obtained from five Orders: sly OIE, . The ser9.3 EID50 per ml and a HA titer of 1,024–2,048 per 25 μl. Electron microscopy revealed that the UPO216 virus particles showed pleomorphic morphology, with a diameter of 80–300 nm. Glycoprotein projections of approximately 10 nm in length were evenly distributed across the envelope of the virus particle. Herringbone-like structures (nucleocapsids) were packaged inside the virion  and APMV-9 (R = 0.125). The higher R-values for APMV-1 and APMV-9 were due to weak cross-reactions in the cross-HI test.The UPO216 virus was successfully propagated in ECEs, and the harvested infective allantoic fluid had a titer of 10KY511044). This length conforms to the “rule of six,” which plays an important role in the replication of paramyxoviruses  showed that UPO216 was closely related with known APMVs, especially with the highest identity with NDV strain 08-004  for N protein gene  and with NDV strain BHG/Sweden/94  for L gene . This indicates that the UPO216 virus might be an APMV.A genome-wide BLASTN search , followed by APMV-9 (56.4%), APMV-12 (52.9%), and APMV-13 (50.9%)  were further tested in HI tests based on antigens derived from other serotypes of APMV, seven  did not cross-react with other APMV serotypes tested, and HI titers for UPO216 ranged from 3log2 to >6log2. The HI titers of the other 13 sera were the same (or higher) for APMV-1 as for UPO216.The 880 serum samples examined herein were first tested for antibodies specific for UPO216 in the HI test. Of these, 20 sera reacted with UPO216  with the potential to cause infectious diseases in poultry. In recent years, several novel APMVs belonging to APMV serotypes 10–14 were reported in wild birds and penguins around the world  from a wild bird fecal sample of unknown species during the 2013/2014 winter season (January 2014) at the UPO wetlands, one of the largest inland wetlands in South Korea. This wetland receives thousands of overwintering migratory birds (mainly ducks and geese) from Arctic regions, starting from late fall. Electron microscopy identified UPO216 as a paramyxovirus, characterized by pleomorphic enveloped virions, projections on the viral envelope, and a “herring-bone” nucleocapsid. Fortunately, UPO216 is unlikely to cause clinical disease in chickens since R = 0.088) and APMV-9 (R = 0.125). Such weak cross-reactivity between APMVs is not uncommon ; Terregino et al., Notably, the UPO216 virus in this study has biologically and genetically unique characteristics distinct from known serotypes of APMV reported so far. First, the cross-HI test showed that UPO216 is antigenically distinct from other known serotypes of APMV, although it did cross-react weakly with APMV-1  belonging to the Order Anseriformes, although the frequency of sero-positivity in wild ducks was relatively low . The majority of UPO216-reactive sera (13/20) from wild ducks (particularly mandarin duck) also reacted with APMV-1 (NDV), which showed HI titers that were the same or greater than those of UPO216, indicating that sero-positive results could be due to mixed infection with APMV-1 and the UPO216 virus, or to a cross-reaction with APMV-1. UPO216-specific antibodies  were detected in serum samples (n = 7) from four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). In particular, UPO216-specific antibodies with a HI titer of 5log2 or greater were detected in mallard ducks. Taken together, these results suggest that at least five species of wild duck  belonging to the Order Anseriformes might be the natural reservoir for the UPO216 virus in Far Eastern countries, including Korea. Further virus isolation and/or serological surveillance studies in wild ducks, especially migratory birds, are needed if we are to better understand the genetic diversity and ecology of the UPO216 virus.The species of wild birds from which the UPO216 virus was originally isolated was not identified at the time of sampling, since virus isolation was conducted from fresh duck droppings in the UPO wetlands. To identify wild bird species that may act as a natural reservoir for the UPO216 virus, we conducted serological surveillance of a variety of wild birds in Korea, including Conceived and designed the experiments: HJL, JK, KC; Performed the experiments: HJL, JK, EL, BS; Supervised and discussed the experiments and data: KC, YL, HSL; Wrote the manuscript: HJL and KC.This research was supported by a grant from the QIA (no. N-1543084-2015-99-02), Republic of Korea. The funders played no role in the study design, data collection and analysis, the decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zika virus (ZIKV), was widely reported in Latin America and has been associated with neuropathologies, as microcephaly, but only few seroprevalence studies have been published to date. Our objective was to determine the seroprevalence amongst Bolivian blood donors and estimate the future potential circulation of the virus.A ZIKV seroprevalence study was conducted between December 2016 and April 2017 in 814 asymptomatic Bolivian volunteer blood donors residing in various eco-environments corresponding to contrasting entomological activities. It was based on detection of IgG to ZIKV using NS1 ELISA screening, followed by a seroneutralisation test in case of positive or equivocal ELISA result.Aedes aegypti activity and the virus spread was not limited by previous immunity to dengue. Cases were geo-localised in a wide range of urban areas in Santa Cruz and Trinidad. No differences in seroprevalence related to gender or age-groups could be identified. It is concluded that ZIKV has been intensely circulating in the Beni region and has still a significant potential for propagating in the area of Santa Cruz.Analysis revealed that ZIKV circulation occurred in tropical areas  but not in highlands . It was modulated by  Aedes mosquitoes, and related to dengue and yellow fever virus. It was originally believed to be responsible for a mild febrile illness in Africa and South-east Asia. However, in recent years, ZIKV has been responsible for outbreaks in the Pacific Islands before massively spreading in Latin America and the Caribbean. On this occasion, ZIKV has unexpectedly been associated with non-vector transmission  and with severe complications such as foetal abnormalities  and Guillain-Barré syndromes.Zika virus (ZIKV) is a virus of African origin, transmitted by Aedes aegypti populations makes plausible further spreading of the disease.Little is known about the actual proportion of the populations infected by ZIKV in Latin America. Here, we report a seroprevalence data in this region, after studying 814 asymptomatic Bolivian volunteer blood donors residing in various eco-environments corresponding to contrasting entomological activities. We conclude that ZIKV has been circulating in Bolivian tropical areas but not in highlands, and that the epidemic has not been limited by previous immunity against dengue. Specific attention should be paid to the region of Santa Cruz, where the seroprevalence is still limited, but the density of  Flavivirus transmitted to humans mainly by Aedes mosquitoes. It has been responsible over the last decade for outbreaks in Pacific Islands [e.g., 2014 in Haiti [Zika virus (ZIKV) is an arthropod-borne  Islands  , in Lat Islands  or early Islands  in Brazi Islands ), and inin Haiti , 2015 inin Haiti ). The fiin Haiti .th century [ZIKV is of African origin, but an Asian lineage emerged presumably in the first part of the XIX century  and the  century  as well  century  were repAedes aegypti mosquitoes). Few ZIKV seroprevalence results in the region have been made available yet, most probably due to the technical difficulty to distinguish antibodies to ZIKV from cross-reacting antibodies to the other flaviviruses, in particular dengue virus. Specific detection of antibodies to ZIKV can be improved when using demanding seroneutralisation methods for the primary detection of antibodies, or for confirmation of a more convenient screening assay such as an ELISA test.Our capacity to estimate the future spread of ZIKV disease in South America significantly depends on our knowledge of the immune status against ZIKV in the populations exposed to the potential vectors  and chikungunya virus (CHIKV), two arboviruses known to be transmitted by the same vector locally. DENV has been reported in Bolivia since 1931. In 1948, Bolivia was declared  Bolivia , 15. CHIin humans.We conducted a study with the help of five Bolivian regional blood banks, representing a variety of eco-environments : 814 volunteer blood donors from Santa Cruz de la Sierra (n = 200), La Paz (n = 161), Cochabamba (n = 152), Tarija (n = 196), and Beni n = 105) provided before blood donation their oral consent for detection of IgG to ZIKV. All donors accepting to participate in the study and providing consent were considered eligible. No specific sampling was performed and leftovers of blood samples collected and stored at -20°C after completion of laboratory analyses were used. The contribution of each site was evaluated locally according to the actual possibilities to recruit donors, process samples and provide the epidemiological data requested (see below). The sample size was in agreement with previous epidemiological studies of arbovirus seroprevalence in other countries and expected to provide a reliable picture of the global epidemiological situation , 10. Sam5 provideBlood samples and personal data  were irreversibly anonymised.Adults blood donors approved to participate in the study by providing oral consent during the face-to-face questioning before blood gift. This procedure was considered the most suitable by local blood banks. The sampling and analysis protocol was approved by the ethics committee of the Medical College of Santa Cruz.Samples were tested for the presence of IgG to ZIKV as previously described ,17. In bAedes aegypti), we randomly selected approximately half of the samples on each site . They were tested for the presence of IgG to DENV and CHIKV (Euroimmun dengue ELISA IgG and chikungunya ELISA IgG assays) according to the manufacturer's recommendations.To estimate the serological immune background against dengue and chikungunya  and 6 ZIKV ELISA ratio groups .Statistical analyses were performed using IBM-SPSS Statistics v 24.0.0.0 software. Statistical association between ZIKV seropositivity and age-group or gender was evaluated, as well as relationship between ZIKV seropositivity and DENV or CHIKV seropositivity .Zika virus strain H/PF/2013: KJ776791.2Aedes borne viruses ranges from November to April. The high seroprevalence rate in Beni ) is in agreement with the report of an outbreak of febrile cases with rash locally, with laboratory PCR confirmed cases before our study (performed in December 2016). Since cases were reported in Beni until the end of the rainy season, it is likely that the final seroprevalence rate is even higher nowadays in this region.Serological results in the different sites for ZIKV are shown in The significant rate in Santa Cruz ) is in line with the report of PCR confirmed cases locally before the collection of samples (in March and April 2017). Detection of cases benefitted from the local implementation of the Cenetrop National Reference Laboratory, but no clear epidemic pattern was reported. Since the study was performed locally at the end of the rainy season, it is most probable that the seroprevalence rate remained locally at a lower level than in Beni.vs 35.2%), possibly reflecting the frequent and intense immune stimulation against ZIKV in the Beni population.Of note, the proportion of ELISA positives confirmed by VNT was much higher in Beni than in Santa Cruz . For chikungunya, approximately half of the population is seropositive in the tropical regions and less than 10% in highlands. The differences observed with dengue most probably reflect the lower number of epidemic waves of chikungunya that hit the country. Dengue was first reported in Bolivia in 1931 and since then has been circulating intensely with major epidemics in the tropical regions[Aedes aegypti-borne viruses in Bolivia, in particular that of the recently introduced CHIKV.l regions and, ovel regions, limitin−12), but also to CHIKV (p<10−15), obviously pointing to exposure to a common risk factor: the bite of the Aedes aegypti vector of the three diseases.This study has classical limitations linked to the sampling procedure in blood donors . However, many previous studies on arboviruses including dengue virus chikunguA. aegypti in all 5 departments investigated [cases were identified from the city centres to outlying district, reflecting the wide distribution of A. aegypti. According to previous information relating to the circulation of dengue in Bolivia and corroborated by our present seroprevalence data, the spread of Zika virus was not limited by previous herd immunity to dengue virus. However, it remains possible that prior immunity to DENV modified the epidemiological pattern of the virus global spread.We conclude that this seroprevalence study confirms the circulation of Zika virus in Bolivia. Despite previous reports of the presence of stigated , 28, 29,A. aegypti in Martinique. With reference to the Martinique outbreak and seroprevalence study, a minimum seroprevalence rate around 50% seems to be required to provide herd immunity that can stop ZIKV circulation. Accordingly, the ~21% rate observed in Santa Cruz (in the last phase of the arbovirus circulation period) would be insufficient to give protective herd immunity in the presence of abundant potential vectors and intense entomological activity, and with sustained circulation and potential reintroduction of the virus in Latin America. It is therefore expected that ZIKV circulation should be limited in the near future in the Beni Region , but, in contrast, ecological and epidemiological conditions are favourable for further circulation of the virus in Santa Cruz, with its consequent complications especially in pregnant women.The same methodological protocol has been used previously to estimate ZIKV seroprevalence in Martinique Island  (CaribbeS1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TablePrevalence between males and females were compared for each site and globally using the chi-square test and were insignificant for p-value at 0.05.(DOCX)Click here for additional data file.


Sergentomyia spp, and subsequently DASHV RNA was detected in a second pool of Phlebotomus papatasi. Genetic and phylogenetic analyses based on complete coding genomic sequences indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], (ii) there is a common ancestor to DASHV, Sandfly fever Sicilian- (SFS) and SFS-like viruses isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV, SFCV and SFTV except those isolated in Iran which forms sublineage Ib (DASHV). Accordingly, we suggest to approve Sandfly fever Sicilian virus species consisting of the all aforementioned viruses. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran.Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. Based on morphological identification, they were sorted into 315 pools according to species, sex, trapping station and date of capture. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated from one pool of  Sandfly fever Sicilian virus species. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran.Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated / detected in two pools. DASHV strain was isolated in cell culture and complete genome sequence was determined. Sequence analysis indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], a virus that is known to cause self-resolutive incapacitating febrile illness in humans, (ii) there is a common ancestor to DASHV and all other variants of SFSV isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV strains, whereas Iranian viruses are most closely related and should be individualized as DASHV (sublineage Ib). Although discovered first in the 1940's, SFSV is still listed as "tentative species" by the International Committee for Taxonomy of Viruses. Based on the results described in this study, we propose to approve  Phlebovirus, family Phenuiviridae currently contains 9 recognised virus species (grouping 37 viruses), and 33 tentative species  and Corfou [CFUV]) are listed in the IXth report of the International Classification for Taxonomy of Viruses (ICTV) ) and reverse primers (DASHV -N-REV [TCCATTGCACTCCCAGGAAC]) were combined with the fluorogenic TaqMan probe (DASHV -N-Probe [6FAM-TGGACTGTCCAAGCTGTGGAGG-TAMRA]), and used with the Go Taq Probe 1-Step RT-qPCR (Promega) as previously reported .2-tissue culture flasks containing Vero cell monolayers in EMEM, enriched with 1% Penicilin Streptomycin, 1% L-Glutamine 200 mM, 1% Kanamycin, and 3% Fungizone and 5mL of fresh EMEM containing 5% fetal bovine serum (FBS) was added after incubation at room temperature for 1 hr. To monitor the development of cytopathic effects the flasks were incubated at 37°C in 5% CO2 atmosphere and examined daily using an inverted microscope.Sandfly homogenates of 50μL were inoculated onto 12.5 cmSupernatant corresponding to passage 2 of DASHV infected Vero cells was used for complete genome characterization using Next Generation Sequencing (NGS) as previously described , and PCRhttp://www.ncbi.nlm.nih.gov/genbank) and were aligned together with DASHV using the CLUSTAL algorithm of the MEGA 6 software [Complete coding regions of the S, M and L segments of phleboviruses were collected from Genbank Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 TableEstimates (%) of evolutionary divergence between sequences of the polymerase (A), Gn glycoprotein (B), Gc glycoprotein (C), nucleocapsid (D), and non-structural (E) genes of the selected phleboviruses and the Dashli virus. The upper-right matrix represents pairwise distances between amino acids alignments. The lower-left matrix represents pairwise distances between nucleotides alignments.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.