An epitaxial growth with sharp interfaces between La0.7Sr0.3MnO3 and Ba0.7Sr0.3TiO3 layers was confirmed by scanning transmission electron microscopy and x-ray diffraction. An unambiguous charge transfer involving an electron transferring from the La0.7Sr0.3MnO3 layers to Ba0.7Sr0.3TiO3 layers  across the interface were resolved by electron energy loss spectra analysis. These observations are attributed to the possible modification in the stereochemistry of the Ti and Mn ions in the interfacial region. The out-of-plane lattice parameter, Curie temperature, and magnetoresistance are strongly affected by the thicknesses of the La0.7Sr0.3MnO3 and Ba0.7Sr0.3TiO3 layers. Huge magnetoresistance subsisting to low temperature was also observed in the La0.7Sr0.3MnO3/Ba0.7Sr0.3TiO3 superlattices. All spectral changes identified at a nanometer scale and their potential effect on the degradation of magnetic and transport properties at a macroscopic level. These findings highlight the importance of dependence on sublayer thickness, illustrating the high degree of tenability in these artificially low-dimensional oxide materials.Artificial superlattices constructed with ferromagnetic La Representative aspects of recent finding include a two-dimensional electron gas with magnetic and superconducting ground states at strong electric insulating interfaces between SrTiO3 and LaAlO35, emergent ferromagnetism at antiferromagnetic interfaces between LaMnO3/SrMnO37, interfacial orbital and spin polarization at the interface of a superconductor YBa2CuO7 and a ferromagnet La0.7Ca0.3MnO39, orbital reconstruction at the interfaces of LaNiO3 and LaAlO311, and enhanced polarization in BaTiO3/SrTiO3/CaTiO3 superlattice13, etc. These results also generated an intense debased on the origin of the unexpected properties at the interfaces. However, the competition between multiple order constants induces a great challenge on our ability to predict the resulting novel electronic phases and functionalities and thus to create artificial materials with meet specific application requirements, and these artificial materials in the form of either composites, superlattices, or multilayers. Recently, the superlattices consisting of alternating ferroelectric and ferromagnetic layers yielded unusual electrical and magnetic transport properties that cannot be obtained in either of their constituents, such as Pr0.85Ca0.15MnO3/Ba0.6Sr0.4TiO3 superlattice14, La0.7Ca0.3MnO3/BaTiO315, La0.75Sr0.25MnO3/Ba0.7Sr0.3TiO3 superlattices16, LaMnO3/SrTiO317, La0.6Sr0.4MnO3/0.7Pb(Mg1/3Nb2/3)O3-0.3(PbTiO3) superlattice19, etc. However, most of the research was devoted to evaluate the relationship between the whole magnetic and transport properties and individual superlattice layers quality, and it was proposed that these unusual electrical and magnetic transport properties are attributed to the ferroelectric spacer layer and the associated magnetoelectric coupling with the ferromagnetic layer19. The microscopic structure of the layers and interfaces  and electric structures  should play crucial roles. One the other hand, oxygen vacancies seem to play a key role in these structures, but the puzzle is far from being understood20.Controlled interfaces between complex oxide systems with the perovskite oxides, lead to a variety of novel and unexpected electronic and magnetic phenomena and functionalities at the interface that are absent in the respective bulk materials0.7Sr0.3MnO3 exhibits fantastic rich variety of attractive properties, such as colossal magnetoresistance (MR), a Curie temperature above room temperature (TC ~ 360 K), half-metallicity, high conductivity, and low coercive field23. While Ba0.7Sr0.3TiO3 as good a candidate for application in high-density dynamic random access memories and microwave devices is due to their high relative dielectric constant (εr) values and tunable εr by applying an electric field24. In this work, we have grown the artificial superlattices consisting with a ferromagnetic La0.7Sr0.3MnO3 (LSMO) layer and ferroelectric Ba0.7Sr0.3TiO3 (BSTO) layer on (100) SrTiO3 (STO) substrates, which are of special relevance in practical dielectric and spintronics devices. The aim in this work is to investigate the crystalline microstructure quality and the electrical structures of the individual layers and interfaces of the LSMO/BSTO superlattices (SLs), and to evaluate their effect on the magnetic and electrical properties and the associated magnetoelectric coupling of the LSMO/BSTO superlattices.Mixed-valence manganite La27. Figure c-axis orientation with a good single phase. The SXRD pattern of the SL [LSMO7/BSTO6]15 (L7B6) around (001) and (002) reflections was exhibited in Fig. The LSMO/BSTO SLs composed of individual LSMO layers with 7 and 14 unit cells (u.c.) and BSTO layer thickness of 6, 12, and 24 u.c. with a total periodicity of 15 were designed and fabricated. The LSMO layers with 7 and 14 u.c. were chosen because the dead layer of LSMO is just below 7 u.c.c of these SLs, together with the bilayers and films, were calculated and shown in Fig. 28 and 3.972 Å29, respectively, and that of the STO substrate is 3.905 Å. The lattice mismatch is −0.74% between LSMO and STO, 1.72% between BSTO and STO, and 2.48% between BSTO and LSMO, respectively. Therefore, the LSMO layers and film experience compressive stress along out-of-plane direction, and the BSTO layers and film experience the tensile stress along out-of-plane direction, indicating by red arrows in Fig. H line with the substrate peak, indicating that the superlattices are under the fully coherent strain.From the position of the fundamental diffraction peak of multilayers, the out-of-plane lattice constant LSMO) and BSTO (tBSTO) layers, i.e., the out-of-plane lattice constants of the LSMO/BSTO superlattices slightly decrease with increasing the thickness ratio of tLSMO/tBSTO, or to say that an enhanced out-of-plane lattice constants for a superlattice are obtained with thicker BSTO layers or thinner LSMO layers.The out-of-plane lattice constant of the BSTO film is as large as 4.169 Å, a little larger than that (4.145 Å) of BSTO in LSMO/BSTO/STO bilayer. Both the out-of-plane lattice constants of the BSTO film and the BSTO layer in the LSMO/BSTO/STO bilayer are larger than that of the bulk BSTO due to the tensile stress along the out-of-plane direction. In contrast, the out-of-plane lattice constants of the LSMO film and the LSMO layer in LSMO/BSTO/STO bilayer are both smaller than that of the bulk LSMO due to the compressive stress in along the out-of-plane direction. The out-of-plane lattice constants of the LSMO/BSTO superlattice are determined by the out-of-plane cumulative stress . The out-of-plane lattice constants of the LSMO/BSTO superlattices are among 3.917 and 3.986 Å, tuned by both the thicknesses of the LSMO  using a high-angle annular dark-field (HAADF) detector in combination with electron energy loss spectroscopy (EELS). Figure 34. EELS under STEM mode with high spatial resolution was used to examine the each element of individual atomic columns. EEL spectrum imaging was used to ascertain the interface structure and to probe any chemical disorder . The Ti-L2,3, Mn-L2,3, Ba-M4,5, and La- M4,5 edges can be simultaneously recorded in a line scan across the interface of L7B6. Figure L2,3, Mn-L2,3, Ba-M4,5, and La- M4,5 edges across the STO substrate, three layers of 6-u.c.-thick BSTO, and two layers of 7-u.c.-thick LSMO. From the EELS spectral maps it can be concluded that the interfaces are very sharp and little chemical interdiffusion is present. EELS line scans were used to characterize not only the cations content profiles but also the local Mn and Ti valence along the thickness of LSMO/BSTO superlattices. Figure L2,3 edges from the layer of 7-u.c.-thick LSMO. The spectra of the Mn L edges do not exhibit strong outstanding features as one might expect for these spectroscopic features dominated by multiplet effects31. The line shape of the spectrum depends strongly on the multiplet structure given by the Mn 3d-3d and 2p-3d Coulomb and exchange interactions, as well as by the local crystal fields and the hybridization with the O 2p ligands. It can be seen from Fig. L-edge fine structures among the Mn-O planes of the LSMO layer can be resolved, which is generally expected when the local Mn valence changes35. No obvious chemical shift of the Mn L3 peak was also observed in previous work on LaMnO3-SrMnO3 superlattice samples37. It was proposed if the Mn concentration was constant in their LMO-SMO superlattice, the lack of a chemical shift has been suggested to result from either oxygen vacancies in the LSMO film or the covalent Mn-O bond rather than a purely ionic bond38. An additional peak about 1.5 eV below the main L3 peak can be seen, which is as a signature of Mn4+ ion39. It can be noted that the additional peaks (indicated by the black arrow) of the end interfacial MnO2 planes show a slight enhancement, which can be ascribed to a slightly increased concentration of Mn4+ ion in the two end interfacial MnO2 planes.The identification of the oxidation states of cations at atomic resolution combined with STEM has become a useful tool to identify the charge states in recent years4+, the ratio of the integrated intensity of the Mn L3 and L2 lines (L23 ratio) has been used to quantify the Mn valence state39. The Mn EELS data close to the LSMO/BSTO interface is shown in Fig. L3 and L2 lines are integrated, and their ratio (L23 ratio) is calculated. The calculated L23 ratio is about 2.64. The nominal Mn oxidation state for our La0.7Sr0.3MnO3 is +3.3. According the Varela’s results, the L23 ratio of the nominal Mn oxidation state for +3.3 is about 2.5, and the larger the Mn L23 ratio, the smaller the Mn oxidation state is. The smaller Mn oxidation state should be attributed to the oxygen vacancies present in the LSMO layers. Figure L23 ratio with the distance of LSMO layer from the interface. It can be seen from Fig. L23 ratio close to the interfaces are a little smaller than that inside MnO2 layers, indicating the Mn valence state close to the interfaces is a little higher than that inside MnO2 layers.For investigating the variation of the valence state of Mn, except for the change of the intensity of the shoulder at higher energy corresponding to MnL2,3 edge obtained from line scan cross the 6-u.c.-thick BSTO layer of are shown in Fig. d orbital for Ti4+ while there is one d electron in d orbital for Ti3+, and the EELS spectra are quite different for Ti4+ and Ti3+ with large shift and different line shapes, The reference EELS spectra for Ti4+ and Ti3+ are taken from the SrTiO3 and LaTiO3. It can be seen from Fig. L2,3 EELS spectroscopic features are the mixture of those from Ti4+ (black dashed lines) and from Ti3+ (black arrows), indicating that the Ti sites exhibit mixed valence between 3+ and 4+. The spectral shape of the Ti EELS changes very tiny across the entire layers, unlike the case of the Mn EELS. This difference maybe come from the different mechanism of the electrons transferring between the tg20/t2g1 orbitals of the Ti4+/Ti3+ ions and the t2g3eg0/t2g3eg1 orbitals of the Mn4+/Mn3+ ions, and the t2g0/t2g1 orbital configuration is more efficient electron transfer. The presence of Ti3+ features at interface should be caused by an electron transfer to Ti4+ ion across the interfaces, since the valence of Ti in the stoichiometric BSTO is +4. Such charge-transfer mechanisms across the oxide interface were also occurred at the other oxide interfaces of the heterostructures and superlattices evidenced by the STEM-EELS spectra, and the typically range is within ~1 nm of the interface36. Combination of the EELS analysis of Mn and Ti, an unambiguous charge transfer involving an electron transferring from the LSMO layers to BSTO layers  across the interface are confirmed. Finally, both the oxidization states of the Mn and Ti ions should be changed due to the existence of the oxygen vacancies. The existence of the oxygen vacancies in the LSMO layers were confirmed by the analysis of the EELS data, as shown in Fig. Individual EELS spectra of Ti-2/La(Sr)O/TiO2/Ba(Sr)O and TiO2/Ba(Sr)O/MnO2/La(Sr)O, respectively.Moreover, from the results of HR-STEM and EELS Figs  it can bM(T)). The magnetization is normalized by the volume of the LSMO layer. Magnetization was measured at field-cooled mode in a magnetic field of 0.01 T. It can be seen from Fig. TC), the saturated magnetization moment (MS) under 1 T at 10 K, and corrective field (HC) with the thicknesses of the LSMO layer in the LSMO/BSTO SLs. It can be seen from Fig. M(T) curves of LSMO/BSTO SLs can be divided into two groups according to the magnitude of magnetization and TC. One group includes the LSMO/BSTO SLs with the LSMO layer thickness of 7 monolayers (MLs), of which the saturated magnetization moment (MS) under 1 T at 10 K is about 0.05–0.09 μB/Mn and TC is about 60–70 K. The other are the LSMO/BSTO SLs with the LSMO layer thickness of 14 MLs, of which MS is about twice of the group one (at under 1 T at 10 K about 0.18–0.22 μB/Mn) and TC among 110–180 K.Charge distribution at the interfaces of the LSMO/BSTO SLs could be expected to have a large impact on the transport and magnetic properties of the LSMO/BSTO SLs. In order to understand these, transport and magnetic measurements were performed using PPMS. Figure 40 as well as LSMO films with oxygen vacancies grown under low oxygen pressures41. Since the thickness of the LSMO film is 130 nm, the oxygen vacancies should play the key role in the strong depression of the double-exchange magnetotransport properties in the LSMO film. The SLs of L14B24, L7B12, and L7B6 exhibit similar insulating behaviors as the LSMO film across the entire measurable temperature range, as shown in Fig. The resistivity, ρ, of each superlattice is calculated from its resistance using the simplified assumption that the current flows only in the LSMO layers since the resistivity of BSTO is orders of magnitude higher than that of LSMO and the entire stack rather than only the LSMO layers contribute to the conduction. The temperature dependence of the resistivity of the superlattices and the LSMO film under the magnetic fields of 0, 0.5, 1, 3, 5 T are presented in Fig. 1−xSrxMnO3 which is attributed to the weak localization of carriers due to disorder42, and in ultrathin LSMO films which is attributed to the presence of quantum interference effects due to weak localization43. SL L14B12 also displays similar features, as shown in Fig. Quite different from other LSMO/BSTO SLs, SL L14B6 displays a transition from semiconducting to metallic behavior at 160 K, followed by an increase for temperatures below 40 K, as shown in Fig. TC, because the local moments within each magnetic domain orientate rather unanimously and the suppression of the local moment fluctuations by a magnetic field can be neglected42. In additional, an anomalous positive MR is observed both in SLs L14B6 and L14B12 at low temperature and under low magnetic fields, seen in the inset of Fig. Magnetoresistance, MR = [ρ(H)−ρ(0)]/ρ(H) × 100, as a function of temperature for the LSMO/BSTO SLs and the LSMO film are shown in Fig. LSMO) and BSTO (tBSTO) layers, i.e., the out-of-plane lattice constants of the LSMO/BSTO superlattices slightly decrease with increasing the thickness ratio of tLSMO/tBSTO, or to say that an enhanced out-of-plane lattice constants for a superlattice are obtained with thicker BSTO layers or thinner LSMO layers. On the other hand, an unambiguous charge transfer involving an electron transferring from the LSMO layers to BSTO layers  across the interface are confirmed by EELS. All above observed phenomena indicate that the interfacial effects play a crucial role on the structural and electronic properties of the LSMO/BSTO SLs.The lattice parameters of the LSMO/BSTO SLs are determined by the interfacial strain, tuned by both the thicknesses of the LSMO  across the interface. Another is a preferential out-of-plane 3d eg (3z2−r2) Mn orbital occupation, attributed to symmetry breaking at the interfaces48.The oxygen vacancies always play a key role in the strong depression of the double-exchange magnetotransport properties in the manganite oxides49. The electric field can be generated by ferroelectric BSTO layers in the superlattice structure by producing the charges at the interface, which in turn changes the resistance of LSMO layer. Usually negative colossal MR is observed in manganite bulks and thin films arising from the magnetic field suppressing the scattering of carriers by localized magnetic moments42. Similar phenomenon was also been observed in La0.7Pb0.3MnO3 bulk48 and in the La0.9Sr0.1MnO3/SrNb0.01Ti0.09O3 heterostructure52, which could be attributed to quantum interference effects due to the Coulomb interactions between carriers enhanced by disorder and the interface effect. The charge and spin states are reconstructed at the interfaces in our system and hence affect the electronic and magnetic properties of the entire system, which should be affected the observed positive MR.The magnetoelectric coupling associated with the multilayers of magnetostrictive and piezoelectric perovskite oxides could attribute to these effects3+→Mn4+; Ti4+→Ti3+) across the interface were resolved by EELS. These observations are attributed to the possible modification in the stereochemistry of the Ti and Mn ions in the interfacial region. The out-of-plane lattice parameter, Curie temperature, and magnetoresistance are strongly affected by sublayer thickness. Huge magnetoresistance subsisting to low temperature is also observed in the LSMO/BSTO superlattices. All spectral changes identified at a nanometer scale and their potential effect on the degradation of magnetic and transport properties at a macroscopic level. These findings highlight the importance of dependence on sublayer thickness and temperature, illustrating the high degree of tenability in these artificially layered materials.In summary, an epitaxial growth with sharp interfaces between LSMO and BSTO layers was confirmed by STEM and SXRD. An unambiguous charge transfer involving an electron transferring from the LSMO layers to BSTO layers  at oxygen partial pressure of 5 Pa, which represented the best compromise for both obtaining the smooth interfaces and surfaces and high-quality of the LSMO and BSTO layers. The LSMO/BSTO superlattices composed of individual LSMO layers with 7 and 14 u.c. and BSTO layer thickness of 6, 12, and 24 u.c. with a total periodicity of 15 were realized. For comparison, the LSMO and BSTO thin films and LSMO/BSTO bilayer were also grown at the same conditions. RHEED oscillations were observed and were used to calibrate the number of layers grown. After growth, all samples were in-situ annealed for 30 minutes.LSMO/BSTO superlattices were grown on STO single crystals by a laser molecular-beam epitaxy system (Laser-MBE) combined with a reflected high energy electron diffraction (RHEED). The LSMO/BSTO superlattices were deposited at 590 °C using a XeCl excimer laser  at the BL14B1 beam line of Shanghai Synchrotron Radiation Facility (SSRF), using a 1.24 Å X-rays with a Huber 5021 six-axis diffractometry.To characterize the atomic resolution characterization of the LSMO/BSTO superlattices, state-of-the-art sophisticated aberration-corrected STEM using a HAADF detector were utilized. The HAADF imaging was executed using an ARM-200F  STEM operated at 200 kV with CEOS Cs corrector  to cope with the spherical aberration of the probe-forming condenser lens. More detailed chemical composition and bonding was probed on an atomic scale using spatially resolved EELS.ρ(T)). The magnetotransport properties as a function of temperature and magnetic field were measured by a standard four-point probe method using PPMS.Temperature dependences of magnetization, resistivity, and MR of the films and LSMO/BSTO superlattice were performed using a Physical Properties Measurement System . The standard four-point probe method was used to measure the film resistivity versus temperature (Supporting information


Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV–host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus–host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding. Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a severe and highly contagious disease in pigs that is listed by the World Organization for Animal Health (OIE). The disease is distributed in many countries and areas including Asia, Eastern Europe, Russia, and South America . CurrentPestivirus genus of the Flaviviridae family . CSF. CSFPestThis review aims to summarize recent progress in the virus biology and virus–host interactions at the interface of virus replication, and highlight potential mechanisms in the CSFV life cycle. The review concludes with future perspectives and highlights areas that require further understanding.rns, E1, and E2). The glycoproteins are processed from the precursor Erns-E1-E2 by the host signal peptidase. The Erns protein consists of 227 aa with a molecular weight of around 48 kDa, which is glycosylated with carbohydrate moieties at seven glycosylation sites [rns is present as a homodimer (with a molecular mass of about 100 kDa) [rns contains five α helices and seven β strands with a concave and a convex face and is stabilized by four intramolecular disulfide bonds [rns show that the amphipathic α-helix is inserted slightly tilted into the membrane [rns possesses ribonuclease activity, induces lymphocytes apoptosis, and antagonizes the response of type I interferon (IFN) signaling. In addition, the interaction between Erns and membrane-associated heparan sulfate (HS) [rns that attaches to the surface of cells by interacting with HS [The structural components of the CSFV virions include a capsid protein (C) and three envelope glycoproteins (Eon sites . Due to 100 kDa)  and a he100 kDa)  on the vde bonds . In addimembrane . Erns poate (HS)  or laminate (HS)  mediates with HS . N-linked putative glycosylation sites and six cysteine residues. E1 is a type I transmembrane protein with an N-terminal ectodomain and a C-terminal hydrophobic anchor that attaches E1 to the envelope of the virus [The E1 glycoprotein consists of 195 aa with an apparent molecular mass of 33 kDa, which contains three he virus . E1 and he virus ,18.818CPIGWTGVIEC828 and 869CKWGGNWTCV878  is a highly-phosphorylated protein [The 5′- and 3′-NCRs of CSFV, approximately 373 and 228 nucleotides (nt) in length, respectively, form stem-loops at the N- and C-termini of the genome . The NSPlication . NS2, a of NS2-3 ,39. Howeof NS2-3 . As a muof NS2-3 ,42,43. Nof NS2-3 . The strof NS2-3 . NS4B coof NS2-3 . Analysiof NS2-3 . The CSF protein . Similar protein . A recen protein . NS5B is protein . The str protein . It has  protein . The int protein ,54,55.The CSFV genome can be transcribed into negative-strand RNA that can be used as the template to produce the positive-strand RNA. During this process, NS5B binds to the negative-strand RNA to produce more positive-strand RNA copies . MoreoveUnlike cellular mRNA, the CSFV genome lacks 5′-terminal cap structure, and the IRES located in the 5′-NCR can be recognized by the ribosome to initiate translation . NS3 canFlaviviridae. It has been demonstrated that the peptides 129CPIGWTGVIEC139 and 180CKWGGNWTCV189 of the CSFV E2 protein mediate fusion between viral envelope and cellular membrane [During CSFV infection, interactions between the virus and HS/LamR mediate virus attachment ,16. Subsmembrane ,32  or IRF-7 and blocks type I IFN induction [pro has been resolved, and the interaction domain harbors a TRASH motif to recognize the immune factors [pro and IκBα (the inhibitor of NF-κB) may be involved in the modulation of the NF-κB signaling pathway [Viruses have evolutionary evolved strategies to evade host innate immune responses for successful virus replication. To facilitate virus infection, CSFV Nnduction ,77. The  factors . The hos factors . Hemoglo factors . Our rec factors . Another factors . Recentl factors . Further pathway   and leads to a redistribution of HAX-1 from the mitochondria to the endoplasmic reticulum (ER), which might increase cellular resistance to apoptosis [Acute CSF is associated with high fever, leukopenia, thrombocytopenia, and hemorrhages observed in various organs. During the processes of acute CSF, the virus induces aberrant levels of type I IFN and pro-inflammatory mediators causing a so-called cytokine storm ,94. It h in vivo ,97. Howein vitro ,99. As apoptosis , whereaspoptosis . The NS2poptosis . It is pThe eventual outcome of viral infection usually relies on the host response to the virus. The virus life cycle consists of attachment, entry, uncoating, biosynthesis, assembly, and release. Attachment factors serve to bind the virion and thus help to concentrate viruses on the cell surface. These factors include HS and other carbohydrate structures on the cell surface. However, the factors usually cannot activate the downstream signals of the host to mediate virus entry. The entry receptor(s) can trigger conformational changes of the virion, activate host signaling pathways, and promote endocytic internalization. The attachment of CSFV is mediated by the host cellular HS or/and LamR ,16. As Hpro targets IRF3 and IRF7 to block type I IFN production [Virus infection can trigger a series of signaling cascades in host cells. To establish and maintain persistent infection, the CSFV Noduction ,77, NS5Aoduction , and C ioduction . Thus, ioduction . More reoduction . Dissect


Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present. Flaviviridae, genus Flavivirus  of the ArD 41525 Zika virus isolate into the vaginal canal or rectum of 16 adult rhesus or cynomolgus macaques and monitored them for evidence of infection through 28 days postinoculation (DPI). This dose was selected to correspond to high Zika virus RNA load(s) reported in human semen  are susceptible to Zika virus infection by the intravaginal intrarectal routes. We based sample size estimates for the 2 study groups (rhesus and cynomolgus macaques) on historic reports of experimental infections of Zika virus involving NHPs .Research was conducted under an Institutional Animal Care and Use Committee–approved protocol at the United States Army Medical Research Institute for Infectious Diseases . This protocol complied with the Animal Welfare Act, Public Health Service Policy, and other federal statutes and regulations relating to animals and experiments involving animals. The Institute is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to principles stated in the 2011 Guide for the Care and Use of Laboratory Animals, National Research Council  and 4 female cynomolgus macaques from Cambodia , age range 8.5–9.3 years, were individually housed during the intravaginal inoculation experiment. For the intrarectal inoculation experiment, an additional 4 rhesus macaques from China ; and 4 cynomolgus macaques from Cambodia , age range 8.2–11.4 years, were individually housed. All macaques were prescreened and determined to be negative for Zika virus, herpes B virus, simian T-lymphotropic virus 1, simian immunodeficiency virus, simian retrovirus 1/2/3 antibodies, tuberculosis, Aedes africanus mosquitoes collected in eastern Senegal in 1984  and has been sequenced (GenBank accession no. KU955591). We selected the ArD 41525 isolate because of its low passage history and the ancestral nature of the African phylogenetic lineage (>2 phylogenetic lineages (African and Asian), these lineages constitute a single virus serotype (The ArD 41525 Zika virus isolate used in this study was made from a pool of 10 PFU (8.7 log10 copies) of cell-free Zika virus suspended in 2 mL of phosphate-buffered saline (PBS) was connected to the end of the infant feeding tube and slowly administered  was inserted into the vaginal opening. A 3-mL syringe containing 7.0 log10 PFU (8.7 log10 copies) of cell-free Zika virus suspended in 3 mL of PBS was slowly administered , and a 3–5-cm lubricated, size 7FR, infant feeding tube was inserted into the rectum. A 10-mL 0.9% NaCl flush was slowly administered to soften impacted fecal material lining the rectum. After the flush, 7.0 logAfter exposure to virus, we evaluated macaques daily for signs of illness. The following clinical observations were made daily: presence or absence of rash, appearance of joints, ocular evaluation, presence or absence of blood and source, motor function, presence or absence of cough, urine output, condition of stool, and food consumption. Blood collections and physical examinations, including weight and rectal temperature, were conducted under anesthesia at −7, 1–7, 9, 12, 15, 21, and 28 DPI. Physical examinations included presence or absence of rash, capillary refill time, dehydration skin test time, joint evaluation, ocular evaluation, oral evaluation, presence or absence of blood and source, severity of bleeding if present, presence or absence of exudate and source, severity of exudate if present, presence or absence of lymphadenopathy, and lymph node size. Menstruation patterns were not recorded before inoculation. Menstruation was noted during the daily observations (0–28 DPI), but may have occurred on additional days .We used comprehensive metabolic panels to test serum samples collected in 2.5-mL Z Serum Separator Clot Activator VACUETTE Tubes  by using a Piccolo Xpress Chemistry Analyzer and Piccolo General Chemistry 13 Panel . Complete blood counts were performed on whole blood collected in 1.2-mL S-Monovette K3 EDTA Tubes  by using a CELL-DYN 3700 system (Abbott Point of Care).Before the study, macaques were surgically implanted with T27F-1B radio telemetry devices . The Notocord-hem Evolution Software Platform version 4.3.0.47  was used to capture and analyze data. Temperature data points were averaged and statistically filtered to remove noise and signal artifacts to generate a single data point every 30 s.2 for 4–5 days and then fixed with 4% formaldehyde  in PBS for 24 h. After removal of the overlay, cell monolayers were stained with 2% crystal violet (Sigma Aldrich) in 70% methanol (Sigma Aldrich) for 5–10 min at ambient temperature, and excess stain was removed with running water. Plaques were counted, and results were reported as number of PFU/mL. The lower limit of detection was 1.0 log10 PFU/mL.We performed virus titration on confluent Vero cell  monolayers in 6-well plates by plaque assay. Duplicate wells were infected with 0.1-mL aliquots of serial 10-fold diluted virus in growth medium composed of Dulbecco’s modified Eagle medium , supplemented with 50 μg/mL gentamicin , 1.0 mmol/L sodium pyruvate, 1% vol/vol nonessential amino acids , and 0.4 mL of growth medium. Virus was absorbed for 1 h at 37°C and was then removed before overlaying the cell monolayers with 3 mL of 1% wt/vol Sea-Plaque agarose  in growth medium. Cells were incubated at 37°C in an atmosphere of 5% COg for 15 min at 4°C. A total of 400 μL of the aqueous phase was collected, and the RNA was precipitated by adding 1 μL of GlycoBlue (15 μg/μL) (Ambion) and 400 μL of isopropanol (Sigma Aldrich). Samples were incubated at ambient temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4°C. The resulting pellet was then washed in 1 mL of 75% ethanol (Sigma Aldrich) and centrifuged at 12,000 × g for 5 min at 4°C, after which the pellet was air-dried for 10 min at ambient temperature and resuspended in 50 μL of diethylpyrocarbonate-treated water. Virus RNA was quantified by using a CFX96 Touch Real-Time PCR Detection System  and primers and a probe specific for the envelope gene (bases 1188–1316)  was added to 200 μL of diethylpyrocarbonate-treated water , which was then added to 750 μL of TRIzol LS Reagent (Ambion). Samples were incubated for 20 min at ambient temperature. After incubation, 200 μL of chloroform (Sigma Aldrich) was added, mixed thoroughly, and incubated for 10 min at ambient temperature. After incubation, samples were centrifuged at 12,000 × 10 PFU/mL of Zika virus, and incubated for 1 h at 37°C in an atmosphere of 5% CO2. Confluent Vero cell monolayers in 6-well plates were inoculated with 100 μL of serum/virus mixture in triplicate. Plates were incubated for 5 days at 37°C in an atmosphere of 5% CO2, fixed, and stained with crystal violet as described above. PRNT80 titers were calculated and expressed as the reciprocal of serum dilution yielding a >80% reduction in the number of plaques. Preexposure serum samples collected from rhesus macaques at −28 DPI and from cynomolgus macaques at −20 DPI showed no neutralization activity for Zika virus, indicating that these animals were not previously exposed to the virus. Postexposure serum samples were screened on 7, 15, 21 and 28 DPI.We performed plaque reduction neutralization tests (PRNTs), considered the standard for clinical diagnosis of past infection, to determine preexposure and postexposure immune responses (10 PFU/mL (7.2 log10 copies/mL) for rhesus macaques and 3.5 log10 PFU/mL (6.8 log10 copies/mL) for cynomolgus macaques  of rhesus macaques and 50% (2/4) cynomolgus macaques had detectable viremias; mean peak titers were 3.8 logmacaques . We dete 1:1,280 . We obse 1:1,280 . Menstru10 PFU/mL (8.0 log10 copies/mL) for rhesus macaques and 4.8 log10 PFU/mL (8.6 log10 copies/mL) for cynomolgus macaques (10 copies/mL) and 7 DPI (6.1 log10 copies/mL). Two cynomolgus macaques  had viremia levels ≥5.0 log10 PFU/mL for 2 days. We detected viremia at 3–7 DPI (mean duration 3.0 d) for rhesus macaques and at 2–6 DPI (mean duration 2.8 d) for cynomolgus macaques and virus RNA in serum at 2–7 DPI for rhesus macaques and 1–12 DPI for cynomolgus macaques. By 15 DPI, all rhesus and cynomolgus macaques had seroconverted , as shown by PRNT80 titers ranging from 1:320 to 1:1,280  of rhesus macaques and 100% (4/4) of cynomolgus macaques had detectable viremias; mean peak titers were 4.8 logmacaques . Althoug 1:1,280 .We observed no overt clinical signs, including pyrexia, joint swelling, weight loss, or decreased appetite, for any of the infected macaques. The telemetry unit in macaque C6 failed during the study. Therefore, we also used rectal temperatures to determine the absence of pyrexia  Figure 1Potential marked increases or decreases in clinical laboratory values lasting >1 day in infected macaques were those for glucose ; blood urea nitrogen ; total protein (R7); alanine aminotransferase ; aspartate aminotransferase ; alkaline phosphatase (C6); total bilirubin (R8); γ-glutamyl transferase (C8); amylase ; leukocytes (R5), erythrocytes ; platelets ; neutrophils ; lymphocytes ; monocytes ; basophils ; and eosinophils   , as well as a mechanism by which Zika virus could be introduced to virus-naive regions and initiate human-to-mosquito transmission. Our findings highlight the need for men living in or traveling from areas to which Zika virus is endemic or epidemic to avoid unprotected sexual intercourse.Additional information on high infection rates for adult macaques after intravaginal or intrarectal inoculation with Zika virus


The discovery of infectious particles that challenge conventional thoughts concerning “what is a virus” has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning “giant viruses”, with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host–virus systems that fall into this “giant” category, demonstrating that this field of inquiry presents great opportunities for future research. In their editorial introduction to the “Giant Viruses” special issue of Virology, Fischer and Condit  stated “Mimivirus, is only ~140 nm in diameter) [Some definitions of giant viruses focus only on genome size with lower limits ranging from undefined  to striniameter) . Indeed,iameter) . Howeveriameter) ,8. WhileDendrolimus punctatus cypovirus 22 (32.75 kbp) [Methanothermus fervidus, 1.2 Mb) and the smallest free-living bacterium  [Using a cutoff of genomic content >200 kb pairs (kbp), ~2.2% 115/5356) of all of the completed virus genomes in NCBI fall within the realm of giants A. To dat.75 kbp) . Perhaps.75 kbp) . This in5/5356 of700 kbp) ,13.Chlorella) have GC contents that are ~21% lower than their host’s nuclear genome. While not a defining feature of all large viruses, this GC difference raises interesting questions concerning the scavenging of nucleotides during the infection cycle. Construction of new viruses is in some cases thought to depend on materials “scavenged” from the host cell, yet in the case of these viruses there would seem to be a discrepancy in terms of what would be available for scavenging. An interest side note to this is that mitochondrial and chloroplast genomes are often observed to have such relative low GC content genomes, similar to these viruses [As with observed ranges in genomic size, there is also a wide range of GC content of these viruses relative to the small eukaryotes they infect . On aver viruses ,15, implAcanthamoeba are internalized via phagocytosis, and it has been shown that this process works less efficiently on smaller (<600 nm) particles [The current size range for giant virus particles varies from our operationally defined ~200 nm to >1500 nm in diameter [articles . Additioarticles .Mollivirus) to unconvincing (Pandoravirus and Pithovirus) homologs to MCP [In addition to a tremendous variation in genome and particle size, giant viruses also have highly diverse morphologies that can be broadly categorized into two groups: ovoid and icosahedral . These ms to MCP ,20,21. IMimivirus and its close relatives  are characterized by proteinaceous fibers anchored to the icosahedron capsid [Mimivirus comes from “Mimicking Microbe” [Phycodnaviridae may also use unique structures to gain access to their host, though their mode of entry is typically by adsorption/injection, as opposed to phagocytosis. For example, the Chlorovirus capsid contains one spike located at a unique vertex of the icosahedral capsid that must be oriented towards the host cell surface to initiate infection [Mimivirus and its relatives utilize a five-pointed vertex called the ‘stargate’ structure that permits the first step in activating infection. Infection is initiated by fusion of the internal viral lipid membrane to the phagosomal membrane [Another mysterious aspect of these giant virus particles are the unique biochemical and morphological features. Virus–host interactions are thought to be facilitated in one of two ways: adsorption to the host cell wall, as is typical of algal host–virus systems , or phagn capsid ,25 that n capsid ,27,28. IMicrobe” ). AdditiMicrobe” , implyinnfection . Similarmembrane ,32, whicmembrane . Whether, which have a lipid envelope outside of the capsid. These viruses include a Phaeocystis globosa virus (PgV-07T) [Micromonas pussila [An anomaly among the giant viruses are several viruses that include EhVPgV-07T)  and seve pussila  which al pussila . This ma pussila . An addi(NCLDVs) (described below) package a variety of proteins inside their capsids encoded by either viral or host genomes that are deployed immediately upon infection. For example, the seven proteomes of giant virus particles currently available (below) contain proteins predicted to combat oxidative stress, presumably because viral infections have been shown to generate Reactive Oxygen Species (ROS) that can inhibit viral replication [Pandoravirus salinus carries one viral-encoded oxidoreductase, as well as three host-derived proteins predicted to combat ROS [P. bursaria chlorella virus-1 (PBCV-1) and the more recently described Megavirus chilensis package homologous Cu-Zn superoxide dismutases [M. chilensis, this protein is remarkable for having the unique ability to fold and incorporate key metallic cofactors without the aid of chaperone proteins [C. roenbergensis virus (CroV) and Mimivirus both package novel sulfhydryl oxidases that may function in the formation of disulfide bonds [The nucleocytoplasmic large DNA viruses lication . Interesmbat ROS . P. burssmutases ,39. In Mde bonds ,41. Thesde bonds ,43,44.Acanthamoeba spp. is that while these are permissive, they may not be the natural hosts. Genomic analyses have been used in an attempt to determine natural hosts. In Mimivirus, most of the genes horizontally transferred from eukaryotes originated from amoeba, indicating amoebae are most likely the natural host of Mimivirus, but alternative hosts are still possible [Acanthocystis turfacea chlorella virus 1 (ATCV-1) from human oropharyngeal samples is intriguing: subsequent analyses have shown consistency between the presence of these viruses and reduced cognitive function in humans and mice [One concern regarding giant virus isolation using possible . Indeed,possible  and the possible . Giant vpossible  and havepossible , indicatand mice .Asfarviridae, Phycodnaviridae, Poxviridae, and Iridoviridae [The NCLDV classification was created to define a monophyletic group of families that, when initially conceived, included oviridae . The ratoviridae ,52,53, ooviridae . A NCLDVoviridae . Howeveroviridae  and poteoviridae ,57, it ioviridae ,59. Theroviridae .Asfarviridae [Iridoviridae and Ascoviridae [Phycodnaviridae [Pandoraviridae. Other families, like Poxviridae [Mimiviridae [Marseilleviridae [Pithoviridae [Megavirales [Poxviridae and Ascoviridae, [Pandoravirus, Pithovirus and Mollivirus. In addition, the Megavirales classification required the capacity to assemble viral factories within the cytoplasm of host cells [Megavirales is not considered a classification by the ICTV.The NCLDV classification is not without its shortcomings. As new members are added to the group, the “nucleocytoplasmic” distinction of replicative strategies becomes less useful due to the increasing diversity of virion production. Many NCLDV families utilize a nucleocytoplasmic route for replication, including rviridae , Iridovioviridae , Phycodnaviridae , and Panxviridae , Mimiviriviridae , Marseileviridae  and Pithoviridae , begin aavirales ,64 basedviridae,  as well st cells ,67,68,69st cells  but not Phycodnaviridae, Poxviridae, Asfarviridae, Ascoviridae, Iridoviridae, Mimiviridae, Marseilleviridae, Pandoraviridae, Pithovirus, and Mollivirus) infect a broad spectrum of hosts. In keeping with the NCLDV group’s high degree of variability regarding particle size and host range, these viruses also display varying degrees of reliance on host metabolism and machinery, resulting in a limited number of highly conserved or “core” genes . Yet deMimiviridae by targeting the conserved MCP gene in this clade. However, this approach has its limits; the three recently discovered representatives of Pandoravirus lack the major capsid protein and the D5R helicase, as well as a number of other core NCLDV genes [Pandoravirus genomes, calling into question their inclusion in the giant virus clade despite their particle and genome size [Comparative analyses of the genes conserved amongst different giant virus families has historically supported the monophyletic nature of the NCLDV group, and recent efforts to determine the clusters of orthologous groups (COGs) for giant viruses support their monophyly . The conDV genes . Indeed ome size .Mimivirus and Marseillevirus particles contain messenger RNA molecules, making them the only viruses, to date, that contain both types of nucleic acid [Initially viruses were defined by their intrinsic filterability away from cellular life forms ,76,77, aeic acid . Moreoveeic acid ,80. IndeThe discovery of translational machinery (including that mentioned above) encoded in select virus genomes allows for comparisons to traditionally “cellular” functions normally associated with the three domains of life. Sequence alignments comparing multiple genes involved in DNA replication and repair, transcription, and translation shared between cellular organisms and NCLDVs appear to show deeply branching relationships as ancient as the domain Eukarya. It was subsequently hypothesized that giant viruses evolved from a cellular common ancestor belonging to a currently extinct fourth domain of life, unique from Bacteria, Archaea, and Eukarya ,81. SeemMarseilleviridae [Acanthamoeba and Cafeteria roenbergensis, it was hypothesized that smaller viruses may incorporate genetic material from other organisms phagocytosed by the host.These ideas have proven somewhat controversial, as direct sequence comparison of genes conserved among cellular organisms with virus-encoded homologs is problematic. As selective pressures on similar genes within viruses and their hosts are likely different, accelerated sequence divergence in viruses may exaggerate their perceived distance from the derived gene . Subsequeviridae ,84. Indeeviridae . These oeviridae . As someMimivirus from a water cooling tower [While surveys are not yet exhaustive, giant viruses appear to be found in all environments. Since the discovery of ng tower , giant vng tower , in adding tower . Lastly,ng tower , arthropng tower ,88.Chlorella viruses are much more abundant when their hosts, normally sequestered as endosymbionts of Paramecium bursaria, are made available as a consequence of predatory activity on the Paramecium [Phycodnaviridae-related sequences were found to be highest in abundance, followed by Mimiviridae [Mollivirus particles in permafrost suggests that giant viruses can survive, under the correct conditions, for long periods of time [A powerful tool in the identification of putative new viruses are environmental metagenomic studies , though ramecium . Regardliviridae ,91. It i of time . When co of time  or infec of time , these nHeterosigma akashiwo virus (HaV) [To date, much of the focus on giant viruses has been on their genomics rather than their influence on the environments in which they persist. Several large, dsDNA viruses including EhV , PgV 9898, AaV ,97 and Hus (HaV)  are assous (HaV) . Algal bus (HaV) ,102. WitMimivirus was documented  as a screen [Asfarviridae family.A recent estimate suggested that giant viruses available in culture were infectious to at least 22 different algal species . Globallcumented . In the a screen : unique Cafeteria roenbergensis [. Given this organism is a grazer of primary producers it is possible that infection of this organism by CroV could have effects on lower trophic level organisms. It has been shown that grazing can be an important driver of algal bloom decline [And while it is obvious that there is a dearth of knowledge concerning giant viruses that infect algae in the environment, there is an even larger knowledge gap regarding giant viruses infecting heterotrophic eukaryotes. The most studied of these viruses is CroV, which infects the heterotrophic grazer ergensis . Given t decline , so it sOnly recently have we come to appreciate the possibility of gene transfer between giant viruses and their hosts. A large proportion of giant virus genes comes from diverse sources, including from their eukaryotic hosts . In EhV,Aureococcus) homologs, [Bigelowiella natans, a Cryptomonad algae [Hydra magnipapillata) genome [H. magnipapillata was putatively identified to be of viral origin [Mimiviridae family member. Giant virus particles and marker genes have also recently also been observed associated with zooxanthellae from the genus Symbiodinium, a dinoflagellate typically found closely associated with corals [Physcomitrella patens and Selaginella moellendorffii [A genome wide phylogenetic study of AaV identified a number of genes having their highest phylogenetic affinity to host  genome . Remarkal origin . Major ch corals . Giant vh corals  and somendorffii . The rolEmiliania huxleyi [E. huxleyi are susceptible to EhV infection, while the haploid stage is ‘invisible’ to infection. It has been suggested that during the decline of the Brown tide blooms, a virus-resistant population of the Aureococcus persists, maintaining a relatively high abundance of Aureococcus even after the demise of the bloom [Host–virus interactions result in an evolutionary arms race—leading to the emergence of new diversity in the host and virus population . Hosts o huxleyi . The diphe bloom ,97.Mamavirus within Acanthamoeba castellanii. Because of this, Sputnik only replicates within A. castellanii co-infected with Mamavirus. Infection by the virophage causes abnormal capsid structure of Mamavirus, increasing capsid size and causing abnormal fiber localization on its surface, suggesting a parasitic relationship between the two [Mamavirus decreased infective Mamavirus particle titers by approximately 70% and increased the survival rate of the A. castellanii [Mimivirus appear to have developed a CRISPR-CAS-like system suggested to combat these virophages, called the Mimivirus virophage resistant element (MIMIVIRE) [Mimivirus [. Other interpretations, however, are questioning these conclusions [. Much has yet to be learned in these systems, but virophages may act like both ‘provirophage’ and ‘provirus’, depending on the genomic context at multiple levels.Another interesting characteristic of some giant viruses is their susceptibility to infection by other bioactive particles, termed “virophage”. The first virophage to be isolated was named Sputnik , which r the two . Co-incutellanii . Similartellanii ,141,142.tellanii . Some clIMIVIRE) . Interesimivirus ,145. Othclusions . Much haMimivirus has driven both the nascence and evolution of a new area of scientific inquiry. Giant viruses are now the topics of evolutionary, ecological and biotechnological inquiries. Moreover, broad-scale efforts to identify new virus–host systems, ranging from classic culture-based approaches to newer bioinformatics efforts to link viruses and their hosts [The discovery of ir hosts  will soo


OVATE gene encodes, are plant-specific transcription factors family. Some members of OFPs have been shown to function as transcription factors to regulate plant growth and development, but little is known about the function of AtOFP8.Transcription factors could regulate multiple aspects of plants growth and development, which is significant to plants. Ovate family proteins (OFPs) that are named due to contain OVATE domain, a 70-AA C-terminal conserved domain from the protein Arabidopsis thaliana. First, we observed that the distribution of epicuticular waxes of wild type plants was more than that of Atofp8-1 deletion mutants, but less than that of 35S:HA-AtOFP8 transgenic overexpression lines not only on the leaves but also on the stems utilizing scanning electron microscopes. Second, we extracted waxes from leaves and stems of three types of plants respectively to measure the waxes content and composition by gas chromatography–mass spectrometer (GC–MS), and the results of the total content of waxes were consistent with the results of scanning electron microscopes. Finally, we found that the expression of 12 genes related to the synthesis and metabolism of waxes was changed in the Atofp8-1 mutants and 35S:HA-AtOFP8 transgenic lines compared with wild type plants.Here, we found AtOFP8 maybe involve in transcriptional regulation of the epicuticular waxes in These findings suggest that AtOFP8 could change the accumulation of epicuticular waxes. OVATE gene encodes, are plant-specific family of regulatory proteins  have been proved to be a novel transcriptional factor family, which could control multiple aspects of plant growth and development , a gene encoding the key enzyme in gibberellin (GA) biosynthesis  complex during early embryo sac development  that are named due to contain OVATE domain, a 70-AA C-terminal conserved domain from the protein LTP4, a gene of lipid transfer proteins gene family, could bind to fatty acids and transfer phospholipids between the membranes  is responsible for the synthesis of primary alcohols, which involves in epicuticular waxes production  and their derivatives, which are significant to plants  was used as wild-type plants in our experiment. 35S:HA-AtOFP8 transgenic lines and Atofp8-1 mutants were previously reported  were prepared with an ultramicrotome. After staining, the sections were observed under a transmission electron microscope.The stems and the rosette leaves of 5-week-old plants were chosen to fix in a solution containing 2.5% glutaraldehyde in 0.1 mol L−1) was used as a carrier gas during which the oven temperature was increased from 80 to 290 °C at a rate of 4 °C/min and finally 30 min at 290 °C.150 mg of the stems and the rosette leaves of 5-week-old plants were weight to put into small beakers respectively and 5 mL of chloroform were added into small beakers for 1 min at room temperature. After adding 5 µg of C24 into the extracted chloroform solvent as internal reference, the solvent was transferred into GC bottles and evaporated under a gentle stream of nitrogen. Then 10 µL bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 10 µL pyridine were added into the GC bottles to redissolve waxes samples and the waxes mixtures were heated for 1 h at 70 °C. Samples were measured by injecting 1 µL of sample and helium , an expression microarray data analysis software of this corporation.ACTIN 2 gene. qRT-PCR reactions were performed in biological triplicates using RNA samples extracted from three independent replicate samples. The comparative ∆∆CT method was used for the calculation of the results.RNA of 3-week-old plants leaves was extracted using RNA extraction kit according to the manufacturer’s instructions. Reverse transcription (RT) was performed according to reverse transcription kit instructions to synthesize first-strand cDNA. Quantitative RT-PCR reactions were performed using SYBR green fluorescent quantitation PCR kit. The PCR primers used were listed in Table When the plants grew up to 5 weeks old, 1 g of rosette leaves was weight and put into erlenmeyer flasks wrapped with aluminum foil. Then 30 mL of 80% ethanol were poured into erlenmeyer flasks. Remove 3 mL solution from erlenmeyer flasks to measure absorbance at 647 and 664 nm using a spectrophotometer as the amount of extracted chlorophylls every 10 min. The solution using for measuring was back into erlenmeyer flasks every time. The absorbance measured after 24 h was as the total amount of chlorophylls. The calculation of chlorophyll content was based on the method previously  was calculated by the formula:35S:HA-AtOFP8 transgenic plants and Atofp8-1 mutant were previously reported  represented the ratio of gene expression level in 35S:HA-AtOFP8 transgenic plants to Col-0 in the data analysis. FC (Atofp8-1 vs Col-0) represented the gene in Atofp8-1 mutant and Col-0 expression levels in the ratio. It was generally considered that there was no significant difference in gene expression in the range of 0.5–2.0 for FC values, while genes with FC values greater than 2 or less than 0.5 were considered significant expression differences. There were 300 genes whose FC (35S:HA-AtOFP8 vs Col-0) value was more than 2 or less than 0.5, among which 192 genes were up-regulated, which were mainly related to photosynthesis, glucose metabolism, amino acid metabolism, fatty acid metabolism, alkanes and flavonoids biosynthesis  value more than 2 or less than 0.5, which 268 genes were up-regulated, mainly related to DNA replication, plant hormone signal transduction, glucose metabolism, photosynthesis, alkanes, pyrimidines and organisms alkaloid biosynthesis processes (Fig. To find out which genes caused by the change of epicuticular waxes, we made gene chip analysis among the KCS10 was up-regulated by more than tenfold in 35S:HA-AtOFP8 compared with Col-0. The expression of LTP4, FAR4 and GL2 also increased by more than 5 times in 35S:HA-AtOFP8 (Fig. CER4, ADH1 and At2g18370 was raised about twice in 35S:HA-AtOFP8, while significantly down-regulated in Atofp8-1 compared with Col-0 (Fig. CER2, At2g42990 and At1g06360 in 35S:HA-AtOFP8 was less than half of Col-0, while up-regulated in Atofp8-1 compared with Col-0 (Fig. We chose some genes involved in epicuticular waxes synthesis and metabolism from gene chip results to test by qRT-PCR Table . ResultsFP8 Fig. a. In addl-0 Fig. b. ContraAtofp8-1, but faster than that in 35S:HA-AtOFP8 (Fig. 35S:HA-AtOFP8 after 70 min (Fig. 35S:HA-AtOFP8.After observing so many changes related to epicuticular waxes, we made water loss assays to test whether stratum corneum changed. Results showed that water loss in Col-0 was slower than that in FP8 Fig. a. FurtheAtofp8-1, Col-0 and 35S:HA-AtOFP8. We analyzed microscopic distribution of epicuticular waxes, all kinds of component content, gene expression difference, which all results could demonstrate that AtOFP8 was able to regulate epicuticular waxes accumulation in some way. This laboratory finding provides a new direction for research AtOFP8 even AtOFPs.To explore the function of AtOFP8, we designed this experiment using 35S:HA-AtOFP8 leaves, alkanes were significantly increased compared with the wild type plants, while primary alcohols were significantly reduced (Fig. Atofp8-1 did not change significantly.Although the total amount of waxes in leaves was little difference, changes in specific components were obvious. Maybe AtOFP8 only changed the conversion of some certain components in leaves, so the total amount of waxes in leaves changed little. For example, in the ced Fig. c, d. AnoDue to diverse component of epicuticular waxes and its complex metabolic process, we do not determine what a role AtOFP8 plays in the metabolic process of waxes. In addition, functional redundancy exists in the transcription factors AtOFPs all the time, so it is difficult to find the its specific function invisibly. This is another reason that we cannot understand which part AtOFP8 affects epicuticular waxes accumulation. In spite of these, various aspects of changes of epicuticular waxes are obvious enough to prove that it plays an important role that AtOFP8 could change genes expression to affect epicuticular waxes accumulation.


Drosophila suzukii (Matsumura) is one of the most damaging and costly pests to invade temperate horticultural regions in recent history. Conventional control of this pest is challenging, and an environmentally benign microbial biopesticide is highly desirable. A thorough exploration of the pathogens infecting this pest is not only the first step on the road to the development of an effective biopesticide, but also provides a valuable comparative dataset for the study of viruses in the model family Drosophilidae. Here we use a metatransciptomic approach to identify viruses infecting this fly in both its native (Japanese) and invasive (British and French) ranges. We describe eighteen new RNA viruses, including members of the Picornavirales, Mononegavirales, Bunyavirales, Chuviruses, Nodaviridae, Tombusviridae, Reoviridae, and Nidovirales, and discuss their phylogenetic relationships with previously known viruses. We also detect 18 previously described viruses of other Drosophila species that appear to be associated with D. suzukii in the wild. Drosophila suzukii (Matsamura) is an invasive dipteran pest of soft fruit belonging to the subgenus Sophophora. Unusual within the genus, the larvae are well adapted to feeding on ripe fruit still on the plant, adult females possess a heavily sclerotized saw-like ovipositor that allows oviposition under the skin of ripening fruit, and their olfactory system is adapted to respond to fruit rather than microbe volatiles  and invasive (British and French) ranges. We describe eighteen new RNA viruses, representing ten different virus families, and confirm their presence in RNA pools using reverse transcription PCR (RT-PCR). We place these viruses in the phylogenetic context of recent metatransciptomic studies in the host genus  in 2013 (collection by AX and SF); in Kent, UK  during the late summer of 2014, 2015, and 2016 (N.C.M.); and in three locations across Honshu, Japan, during May 2016 (N.C.M. and M.N.): Tokyo University of Agriculture and Technology, Fuchu ; Naganuma Park, Tokyo ; Shimaminami Shima, Yamagata Prefecture ; Agriculture Total Centre Kaju Research Institute, Fukushima ; and Fuefukigawa Fruit Park, Yamanashi . We used a combination of commercial bait traps with cotton soaked in a proprietary liquid attractant , and a standard sweep net to catch adult flies. Traps, hung at field margin and woodland sites, were collected at intervals of 2–3 days. All individuals were sorted into vials by trap and species within 3 h of collection. We aimed to morphologically identify all species of Drosophila caught . In addition to adult fly samples, larvae were extracted from infested fruit collected in 2016 from UK and Japan with sterile forceps. Although no Drosophila pathogens have previously been reported from the larval stage alone, through their collection we aimed to address the possibility that our sampling method was biased towards mobile adult flies able to respond to attraction based traps.We collected 4450 individual a caught ; however® (Invitrogen), before storage at –80°C. We treated pooled RNA samples for possible DNA contamination using DNase  prior to library preparation. To verify RNA quality we tested for contamination using Qubit® and Nanodrop® spectrophotometers. For flies collected in the UK and Japan, library preparation and strand specific sequencing was performed by Edinburgh Genomics  using Illumina NGS library preparation kits and the Illumina Hi-Seq platform with 120 or 150 nt paired end reads. To increase representation of viral and host protein coding RNAs, all libraries underwent depletion of rRNA using Ribo-Zero rRNA Removal Kit (Illumina). Flies collected in France during 2013 were sequenced separately at Beijing Genomics Institute  using paired-end 90 nt reads using the HiSeq 2000 platform. These libraries underwent Duplex-Specific Thermostable Nuclease normalisation and poly-A selection. This process, although enriching for viruses by rRNA depletion, biases virus discovery towards poly-adenylated genomic products only produced by certain viral taxa . All raw reads have been submitted to the NCBI sequence read archive under project accession PRJNA402011 .We pooled trap catches from within a sampling location and immediately extracted RNA from a subsample of the fly (or larva) homogenate using TRIzolDrosophila, we mapped raw reads against the D. suzukii genome and transcriptome using Bowtie2  coding sequences unless otherwise stated. The RdRp is generally the most conserved protein across RNA viruses, making it suitable for phylogenetic analysis of this diverse set of virus taxa . RdRp geTo confirm the presence of the newly discovered viruses in original RNA pools, we used RT-PCR to screen for short amplicons of each virus’ longest ORF, where possible spanning part of the RdRp gene. We designed primers using the Primer3  plugin f−3, and searching the NCBI ‘nr’ protein database using BLASTp. Only ORFs of 100 amino acids or longer were annotated, unless notable similarity to closely related viruses was evident. ORFs of less than 200 amino acids that were nested completely with larger ORFs were disregarded, unless they displayed high similarity to known proteins.For viruses with complete, or near complete genomes, we were able to infer genome structure and identify protein functional domains by first identifying ORFs and then comparing these to the Conserved Domain Database with an expected value threshold of 5 × 10Drosophila virus genomes (including multiple divergent isolates where they were available), a selection of Drosophila ribosomal sequences, and a short region of cytochrome oxidase 1 (COI) that has discriminatory power between Drosophila species. Sequences were mapped with Bowtie2 , to previously published viruses. Those previously described viruses that were detected in D. suzukii are detailed in In total, we generated approximately 280 million read pairs, ranging from 33 million pairs (UK—2016) to 105 million pairs (France—2013) per library. Our assemblies comprised between 18,431 (Japan—2016) and 56,384 (UK—2015) putative transcript contigs. Among these, we identified eighteen new RNA viruses associated with  suzukii . These vD. suzukii Nora Virus. This virus is sufficiently closely related to the D. melanogaster Nora virus and Drosophilaimmigrans Nora virus that a name outside of this local scheme may cause confusion for future studies. During Phylogenetic analysis, a number of virus-like sequences were identified by BLAST in the public Transcriptome Shotgun Assembly (TSA) database. These have been included in analyses to help improve accuracy of phylogenetic inference, but are not further discussed.We have provisionally named these viruses according to the location from which the hosts were sampled. We have chosen not to include taxonomic or host information in the provisional name of the virus, as these are subject to change as phylogenetic relationships are revised and alternative or additional hosts discovered. The one exception to this rule is Drosophilasubobscura Drosophilnogaster . Teise vd ranges .Drosophila subsilvestris in the UK  to merit description (Bactrocera latifrons and Ceratitis capitata), and can also be found in the transcriptomes of their parasitoid, Fopius arisanus . The other virus we identified from this clade, Mogami virus, is closely related to Shayang fly virus 1, a Chuvirus detected in Chinese Diptera , and are therefore highly likely to represent infections of D. suzukii. The first of these is Brandeis virus (MF953177), the genome of which is reported here for the first time , or viruses with biologically constrained host ranges, such as the Sigma viruses, along with Drosophila A virus, Drosophila C virus, and D. melanogaster Nora virus that were known to be present in D. melanogaster samples run alongside the 2016 D. suzukii samples.The remaining previously published viruses were detected at much lower levels . Some ofrn virus  are possDrosophila virus genomes , showed that all viruses with negative-sense genomes were represented by between 15 per cent and 49 per cent positive-sense reads; viruses with double stranded RNA genomes by 53.3 per cent to 70.8 per cent positive reads; and positive-sense viruses 88.3 per cent to 100 per cent positive reads  and in the UK sample of 2015 0.74 per cent of reads mapped to D. melanogaster. The D. melanogaster reads may represent misidentification or cross-mapping, as the species are quite closely related, but it is more likely that they are the result of contamination across libraries through barcode switching as D. suzukii samples were sequenced in parallel with unrelated drosophilid libraries.The virus composition varied markedly among samples from different times and locations . Six of witching  and otheDrosophila pest D. suzukii in its native and invasive ranges. Alongside eighteen new viruses, not previously described from any organism, we confidently identified a further seven viruses associated with this novel invasive host that had previously been described from other Drosophila species. Some novel viruses were detected solely from the native range of D. suzukii and others from the invasive range, but rarely from both habitats.Here we make a first survey of the viruses associated with the invasive D. suzukii. Although their presence as RNA but not DNA implies that they are not expressed EVEs, it remains possible that some are not truly infections in this fly, but may be contaminants of the surface of the fly or infect a commensal, pathogen, or food organism within the fly’s gut lumen. However, we believe that this is unlikely to be the case for most sequences, as previous studies that additionally used the presence of virus-derived 21 nt short interfering RNAs to demonstrate active replication  is distantly related to any known Drosophila virus, but is closely related to Shayang Fly virus 1  or very closely related to a virus in another related hosts (D. suzukii.Given our focus on an invasive species, the potential for a shift in the virological environment associated with invasion is of particular interest. Theory predicts that organisms may experience a ‘release’ from natural enemies, including pathogens, in their invasive range due to low host densities and founder effects at the invasive edge : However viruses  and may ed hosts  and E. ISupplementary DataClick here for additional data file.


ZIKA virus (ZIKV) poses a severe threat to the world. Recent outbreaks of ZIKV after 2007 along with its quick transmission have made this virus a matter of international concern. The virus shows symptoms that are similar to those caused in the wake of dengue virus (DENV) and other flaviviruses, which makes it difficult to discern the viral infection. Diagnosis is further complicated as the virus cross-reacts with antibodies of other viruses. Currently, molecular diagnosis of the virus is being performed by RT-PCR and IgM-captured enzyme-linked immunosorbent assay (MAC-ELISA). The real brunt of the virus is, however, borne by children and adults alike. Case studies of the ZIKV outbreaks in the French Polynesia and other places have suggested that there is a close link between the ZIKV and Gullian-Barre syndrome (GBS). The GBS has closely followed in areas facing ZIKV outbreaks. Although solid evidence is yet to emerge, clinical data integration has revealed a large number of ZIKV patients having GBS. Moreover, the amniotic fluids, blood cord, and miscarriage tissues of mothers have been detected with ZIKV, which indicates that the virus either gets transferred from mother to fetus or seeks direct entry in the fetus, causing microcephaly and other brain anomalies in the newborn babies. Studies on mice have confirmed the link between the ZIKV infection during pregnancy and microcephaly in babies. Reports have highlighted the sexual transmission of the ZIKV, as it has been detected in the semen and saliva of affected persons. The intensity with which the ZIKA is spreading can collapse the health sector of several countries, which are poor. A comprehensive strategy is a need of an hour to combat this virus so as to prevent its transmission and avert the looming threat. At the same time, more research on the cure of the ZIKV is imperative. ZIKA virus (ZIKV) belongs to the family of flaviviruses  that entFigure 1). Whereas, in the Asia, the sylvatic transmission cycle of ZIKV is yet to be reported .The ZIKV belongs to the family, Flaviviridae, genus Flavivirus. The virus is an arthropod-borne virus or arbovirus. The infectious particle of the virus known as virion is surrounded by a lipid membrane embedded by the viral membrane protein (Protein M), and envelope protein (Protein E). The virions are icosahedral, enveloped and contain a single-stranded, non-segmented RNA genome of the positive strand  that encThe diagnosis of undifferentiated febrile illness is challenging as its relevant symptoms overlap with other common infections . The aduThe diagnosis of ZIKA is primarily being done either by RT-PCR-based tests or neutralization assay where immune globin IgM is detected by IgM-captured enzyme-linked immunosorbent assay (MAC-ELISA) . Viral nA. aegypti viruses by eliminating their breeding sites, application of larvacides, use of mosquito repellents, bed nets, avoiding sleep in the day, and to maintain the green and clean environment  strain, leading to a reduction of the local population of A. aegypti. The GM male mosquitoes can mate with wild type females and thus can eliminate them  from the onslaught of ZIKV infection. The relationship between the GBS in French Polynesia is very alarming and could further exacerbate child healthcare in the world. The rise in microcephaly and GBS in the aftermath of ZIKV outbreaks is a serious matter of concern and requires proper treatment so that the disease does not nip out tulips (children) in the bud. In order to develop an effective strategy against ZIKV, and other flaviviruses, there is a need to systematically identify, and address the loopholes in virus research. Diagnostic assays for the ZIKV are garnering intense interest and there is hope that in the near future promising strategies for the improved diagnostics of ZIKV will translate into therapeutic and preventive tools. Development of animal models for fetus development would further deepen the understanding of the transmission of ZIKV from mother to fetus. The present clinical data must be integrated, and new reliable, affordable molecular diagnostic tests must be developed to cope up with ZIKA endemic. Climate change is also a contributive factor in the transmission of many flaviviruses. New reports have outlined that rising temperature of earth suits the breeding patterns of mosquitoes. Globalization of trade is another route for the transmission of the virus specifically due to the rubber tires of transportation vehicles serving as breeding sites for mosquitoes. For the time being, an accurate vaccine for the treatment of the ZIKV is unavailable, therefore, there is little that can be done to reverse the adverse effects that the virus is having on the babies in the form of microcephaly and GBS. New and efficient ways of vector controlling mechanism must be introduced, and influence of environmental factors on ZIKV emergence should be understood in an elaborate manner. Although herd immunity can slow the transmission rate to some extent, it cannot be a replacement for the appropriate vaccination. Hence, it is imperative that efforts to develop therapeutic tools like a vaccine against ZIKV must continue unabated to develop a cure as soon as possible.The cross-reactivity of serological assays and mild symptoms of ZIKV make it difficult for scientists to gauge it at the preliminary stage. The risk of ZIKV multiplies in the areas where DENV is an epidemic as both belong to the family of flavivirus. The emergence of ZIKV is still an enigma for the scientific community; however, a general trend has been observed across the globe in which transmission of DENV, and chikungunya infections are followed by ZIKV infection . The simZika virus is a threat of international concern that requires immediate attention. Success against this threat can only be achieved by continuing the extensive research regarding this virus to be able to find an appropriate vaccine to tackle with the menace. The relationship, mode of action, and transmission among DENV, chikungunya and ZIKV infections should also be verified. A breakthrough in either of the mentioned viruses could be a milestone in the history of medicine as all of these viruses have a high degree of genetic similarity because they belong to the family of flavivirus. Until then, a comprehensive multidimensional strategy must be employed to strengthen public awareness in this regard and control the spread of the ZIKV by curbing its transmission through sexual intercourse, travel to affected areas and global trade.IR and JBL wrote the paper. VB design the paper. WP and JL did the critical analysis and approved the paper.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.


Dengue fever is an important arboviral disease. The clinical manifestations vary from a mild non-specific febrile syndrome to severe life-threatening illness. The virus can usually be detected in the blood during the early stages of the disease. Dengue virus has also been found in isolated cases in the cerebrospinal fluid, urine, nasopharyngeal sections and saliva. In this report, we describe the isolation of dengue virus from the upper respiratory tract of four confirmed cases of dengue.We reviewed all laboratory reports of the isolation of dengue virus from respiratory specimens at the clinical microbiology laboratory of the Kaohsiung Veterans General Hospital during 2007 to 2015. We then examined the medical records of the cases from whom the virus was isolated to determine their demographic characteristics, family contacts, clinical signs and symptoms, course of illness and laboratory findings.Dengue virus was identified in four patients from a nasopharyngeal or throat culture. Two were classified as group A dengue (dengue without warning signs), one as group B (dengue with warning signs) and one as group C (severe dengue). All had respiratory symptoms. Half had family members with similar respiratory symptoms during the period of their illnesses. All of the patients recovered uneventfully.The isolation of dengue virus from respiratory specimens of patients with cough, rhinorrhea and nasal congestion, although rare, raises the possibility that the virus is capable of transmission by the aerosol route among close contacts. This concept is supported by studies that show that the virus can replicate in cultures of respiratory epithelium and can be transmitted through mucocutaneous exposure to blood from infected patients. However, current evidence is insufficient to prove the hypothesis of transmission through the respiratory route. Further studies will be needed to determine the frequency of respiratory colonization, viable virus titers in respiratory secretions and molecular genetic evidence of transmission among close contacts. Dengue virus is rarely identified in respiratory specimens. We retrospectively identified four patients with dengue fever who had the virus isolated from their nose or throat. All the patients had respiratory signs or symptoms. Half had family members who also had respiratory symptoms. Further studies are needed to evaluate the possibility of respiratory transmission of this virus. Aedes aegypti and less commonly by Aedes albopictus. The World Health Organization (WHO) estimates that up to 100 million infections occur annually [Dengue fever is a mosquito-borne disease caused by dengue virus (DENV). There are four serotypes–DENV-1, DENV-2, DENV-3 and DENV-4. Dengue is widely distributed throughout tropical and sub-tropical areas around the world and imposes great economic burden. It is transmitted by The course of dengue usually go through three phases: febrile phase, critical phase and recovery  phase , 2. FebrTimely and efficient diagnosis is crucial to the management of dengue. Current diagnostic tests enable simple, rapid and sensitive detection of the virus. This is achieved by isolation of the virus or serologic or molecular methods depending on the phase of the disease . NonstruAlthough the early diagnosis of dengue fever is usually based on detection of viremia, there are several reports of DENV in cerebrospinal fluid , saliva This study was approved by the institutional review board of the Kaohsiung Veterans General Hospital. Patient data were anonymized.The study was conducted at the Kaohsiung Veterans General Hospital, in southern Taiwan. We identified all isolations of dengue virus from respiratory specimens obtained from patients admitted to the emergency service, hospital or clinics setting during 2007 to 2015. A respiratory specimen was defined as material obtained from the sputum, nasopharyngeal or pharyngeal secretions, or bronchoalveolar lavage fluid. The medical records of each patient were reviewed to provide demographic data, clinical signs and symptoms, contact history, laboratory findings, clinical course and outcome.2. Observations were made twice a week for 3 weeks at 4x magnification until a cytopathic effect (CPE) was evident.Respiratory specimens were sent to the virology laboratory for identification. They were inoculated into six different cell lines: human lung fibroblast (MRC-5), human rhabdomyosarcoma (RD), human lung adenocarcinoma (A549), African green monkey kidney cell (Vero), Rhesus monkey kidney (MK2), and Madin Darby canine kidney (MDCK). Fresh Eagle minimal essential medium was added and incubated at 35°C with 5% COCPE positive cultures were identified using indirect immunofluorescence assay (IFA) screening kits for respiratory viruses  and enteroviruses  according to the manufacturer’s instructions. Respiratory IFA contains antibodies which recognize adenovirus, influenza A and B, parainfluenza 1, 2 and 3, and respiratory syncytial virus. Enterovirus IFA contains antibodies which recognize echoviruses, coxsackie A and B, polio 1,2, and 3, and enteroviruses 70 and 71.TM One-Step RT-PCR with Platinum Taq kit; Invitrogen Corporation) were performed. The amplification reaction consisted of cDNA synthesis at 50°C for 30 minutes; predenaturation at 95°C for 1 minute, PCR amplification for 45 cycles at 95°C for 30 seconds, at 50°C for 30 seconds, and at 72°C for 7 minutes; followed by a final extension step at 72°C for 7 minutes. PCR products were analyzed by electrophoresis on a 1.5% agarose gel; the expected amplicons were 169 base pair (bp) for DENV-1, 362 bp for DENV-2, 265 bp for DENV-3, and 426 bp for DENV-4.Viral RNA was extracted from each culture supernatant using the QiAmp viral RNA kit (Qiagen inc.) according to the manufacturer’s instructions and analyzed using classic RT-PCR. We used primer DV1 and type-specific DSP1, DSP2, DSP3 and DSP4 according to a previous study . One-steDescriptive statistics were used to characterize the study population, clinical features and laboratory findings.Our virology laboratory received 31893 respiratory specimens during 2007–2015. Dengue virus was isolated from either the nasopharynx or the throat of 4 patients. Their medical records were reviewed and summarized below.9/L, hematocrit (HCT) of 35.9% and a platelet count of 166×109/L. She tested positive for serum dengue NS1 antigen and negative for dengue IgM and IgG (SD BIOLINE Dengue Duo). A quick test (BD VeritorTM System For Rapid Detection of Flu A+B) was positive for influenza A. She was treated with oral oseltamivir and subsequently hospitalized. A nasopharyngeal culture obtained after admission was positive for DENV-2. Her condition gradually improved and she was discharged home after 5 days of hospitalization.A 64-year-old otherwise healthy woman presented to the emergency department with fever of 38.0°C for one day in November 2015. This was associated with generalized myalgia, severe headache, rhinorrhea, nasal congestion, productive cough and gingival bleeding. She had no signs of respiratory distress when seen in the emergency department. Laboratory analysis revealed white blood cell count (WBC) of 3.18×109/L, a HCT of 38.6% and a platelet count of 151×109/L. Throat swab tested negative for influenza quick test and negative for beta-hemolytic streptococcus and nasopharyngeal swab was positive for DENV-2. His stool was negative for adenovirus . He was treated for acute bacterial tonsillitis with oral amoxicillin and followed up in the pediatric clinic 2 days after, with marked improvement of symptoms.A 17-year-old young man presented to the pediatric emergency department with intermittent high-grade fever for two days in August 2015. This was associated with myalgia, headache, rhinorrhea, sore throat, productive cough and abdominal pain. Physical examination showed a congested oropharynx and soft abdomen with hyperactive bowel sounds. Laboratory tests showed a WBC of 3.06×109/L, a platelet count of 139×109/L and HCT of 36.2%. A nasopharyngeal influenza quick test was negative. She was discharged with antipyretics, but returned the next day due to nausea, persistent fever and new-onset bilateral otalgia. Her eardrums were normal without discharge. There was no skin rash. She was treated with amoxicillin-clavulanate and antipyretics. A serologic test at day 4 was positive for dengue NS1 antigen. The throat swab collected at day 5 of fever showed a cytopathic effect. The virus culture was positive for DENV-2 by RT-PCR. She became afebrile on day 6 and gradually improved.A 14-year-old girl presented to the emergency department with fever, chills and fatigue for 3 days in September 2010. She had history of pneumonia and acute otitis media (AOM) when she was in elementary school. Her aunt, living with her, had similar signs and symptoms. She had no recollection of a mosquito bite. Her temperature was 38.9°C. Her conjunctivae and throat were congested. Laboratory investigations showed a WBC of 2.78×109/L, HCT of 41.5% and platelet count of 264×109/L. Chest X-ray was unremarkable. He was admitted to the pediatric service and treated with oseltamivir. His fever and other signs and symptoms persisted for 4 days and were accompanied by lethargy, severe bone pain, generalized skin rashes and one episode of epistaxis. Laboratory data revealed a WBC of 2.08×109/L, a platelet count of 70×109/L and hemoconcentration with HCT of 46.9%. The patient was transferred to the pediatric intensive care unit for monitoring because of signs of severe dengue. A nasopharyngeal influenza quick test was negative. Throat swab and blood virus cultures were obtained at day 4 of illness. DENV-1 was isolated from both specimens. Abdominal sonography and chest-x ray showed no signs of pleural effusion or ascites. The patient was treated supportively and was discharged at day 12 of illness.A 12-year-old boy presented to the pediatric emergency room with a temperature of 40°C for one day in October 2008. This was associated with generalized weakness, myalgia, severe headache and vomiting. His father had similar symptoms and was admitted in our hospital at the same time. The child’s blood pressure was 178/78 mmHg and heart rate was 133/min. He had a congested pharynx with bilateral coarse breath sounds. Laboratory test showed a WBC of 8.49×10Their clinical findings are summarized in Four cases of confirmed dengue fever with isolation of dengue virus from their respiratory tract was presented. Their clinical manifestation ranges from mild illness, which can be managed at outpatient clinics, to severe dengue, which required closely monitoring in ICU setting. All patients had respiratory tract symptoms and half of their family members also reported respiratory symptoms. This suggests the possibility of the respiratory route for transmission of dengue virus. We reviewed the literature for evidence supporting this concept.A. aegypti and A. albopictus [Dengue virus is typically transmitted by means of its vector mosquitos. Humans are its primary amplifying host . The incbopictus , making Dengue virus can also be transmitted from infected blood by transfusions , organ tThere has only been one prior report, to our knowledge, of the isolation of DENV from nasal and pharyngeal swabs of a patient residing in a dengue-endemic area . We now There is strong evidence from work conducted on dengue fever in World War II that dengue virus can be transmitted through nasopharyngeal exposure to infected serum . SeventyThere are other reports of possible mucocutaneous transmission of dengue virus. A health care worker was diagnosed with dengue after she accidentally splashed the infected serum on her face, including her eye, nose and mouth. She had nosebleeds, anorexia and eye pain 10 days afterwards. Serological markers indicated acute dengue infection . AnotherA. Albopictus [Before the availability of polymerase chain reaction, typical viral isolation techniques for dengue virus involved inoculating specimens in C6/36 cells, larva tissue of bopictus . Our virbopictus . These fThe cells routinely used for viral culture of respiratory specimens in our laboratory were designed to detect common respiratory pathogen rather than dengue virus. Once inoculated cells show CPE, we would use screening IFA for respiratory virus and enteroviruses. Coinfections with respiratory virus and enterovirus IFA were excluded since all our specimens tested negative for these pathogens by IFA.Since this is a retrospective study, it has certain limitations. The respiratory secretions of dengue–infected patients were not routinely cultured for viral growth. A review of 281 cases of patients with dengue in our hospital in 2015 shows that only 2 patients had respiratory specimens sent for viral culture. The culture cells we used were not optimal for dengue virus growth and may underestimate dengue infection. Of 31893 specimens received, only 4 tested positive for dengue virus. Testing for dengue PCR from respiratory virus and enterovirus IFA-negative specimens were done only when dengue virus was suspected by the microbiologists according to their own experiences.This report of the isolation of dengue virus from the airway secretions of four patients with respiratory symptoms raises the possibility that it may spread by the aerosol route or by direct contact. This concept is supported by studies which show that the virus can replicate in cultures of respiratory epithelium and can be transmitted through mucocutaneous exposure to blood from infected patient. Current evidences are insufficient to prove the hypothesis of transmission through the respiratory route. Further studies are needed to determine the frequency of respiratory colonization in patients with dengue, viable virus titers in respiratory secretions and molecular genetic evidence of transmission of the same strain among close contacts.S1 Tests(DOCX)Click here for additional data file.