On fragmenting three-dimensionally ordered macroporous (3DOM) structures in a controlled manner, via a process that has been likened to retrosynthesis, hexapod-shaped building blocks can be harvested which possess a mesostructured architecture. The hexapod-shaped perovskite catalyst exhibits excellent low temperature methane oxidation activity . First principle calculations suggest the fractures, which occur at weak joints within the 3DOM architecture, afford a large area of (001) surface that displays a reduced energy barrier for hydrogen abstraction, thereby facilitating methane oxidation.Versatile superstructures composed of nanoparticles have recently been prepared using various disassembly methods. However, little information is known on how the structural disassembly influences the catalytic performance of the materials. Here we show how the disassembly of an ordered porous La Disassembly of three-dimensionally ordered materials generates nanoparticles with new structural and physicochemical properties. Here the authors show a fragmentation strategy applied to a perovskite material leading to nanostructures with improved catalytic activity in the methane combustion. However, the associated high cost and poor thermal stability  of the catalytic elements persist as major challenges. Mixed oxides comprising non-noble metal components seem a more promising alternative, with perovskites having gained particular attention20.6Sr0.4MnO3 (3DOM LSMO) was synthesized with a high surface area and a rich presence of surface oxygen species whereby it exhibited a much better catalytic performance than its one-dimensional (1D) counterpart. Recently, new nanoparticle shaping strategies have been established involving the disassembly of preformed macroscopic architectures by cutting, pasting or merging assembled structures. For example, Fukino et al.4. Upon oxidizing the ferrocene groups, the tubes could be cut into a large, stable rings and then reversibly reassembled into nanotubes by reduction of the ferrocene groups. The findings reported by Fukino et al.It is of great interest to use methane as an alternative fuel to coal and oil due to its high ratio of hydrogen to carbon which leads to comparatively lower greenhouse gas emissions. Catalytic methane oxidation is often employed to stabilize ‘lean' flames (those with a low fuel to air ratio) at relatively low temperatures as compared to non-catalytic combustion, thus preventing the formation of noxious nitrogen oxidesHerein, we implement a rational fragmentation strategy involving a 3DOM architecture as the precursor material, which is disassembled into well-defined structural building units to produce a mesostructured LSMO perovskite catalyst. The disassembly approach is promising for acquiring nanostructures for a wide range of catalytic applications which are otherwise difficult to obtain by other templating methods. We demonstrate the synthesis of 3D-hm LSMO, where h refers to hexapod and m refers to mesostructured. The hexapod building blocks are derived from a highly crystalline mesoporous 3DOM framework, which is an inverse replica of a well-ordered hard template polymethyl methacrylate (PMMA) microsphere array. The surface chemistry and structure of the hexapod perovskite particles were examined by X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) computational studies so as to understand the origin of the enhanced methane oxidation activity exhibited by the 3D-hm LSMO.2 (30 ml per min) at 300 °C for 2 h. Fragmenting the 3DOM framework in a controlled manner was found to be important with the structures comprising a low degree of connectivity points between structural building blocks that were prone to cleavage under specific conditions  after which the polymer microsphere template was thermally removed . The 3DOnditions .An assessment of the structural evolution of PMMA, the 3DOM network, and the 3D-hm LSMO was conducted using field-emission high-resolution scanning electron microscopic (FE-HRSEM) and 3D-environmental atomic force microscopic (3D-eAFM) techniques . Figure d spacing of 0.270 nm which is consistent with the (110) spacing of cubic La0.6Sr0.4MnO3 perovskite, indicating the formation of 3DOM LSMO with good crystallinity (JCPDS PDF No. 04-016-6114). The field-emission high-resolution transmission electron microscopy (FE-HRTEM) technique can provide more complete and compelling detail on the structure and particle morphology. The spherical elements of self-assembled monodisperse PMMA microspheres are interconnected and regularly oriented, forming a single-cubic crystal of 3DOM LSMO with L-lysine as the stabilizing surfactant . Intenti2 adsorption–desorption isotherms of the 3D-hm and 3DOM LSMO samples show similar type IV isotherms (p/p0=0.9–1.0) and H2- (p/p0=0.2–0.6) type hysteresis loops, demonstrating the retention of macroporosity and mesoporosity within in the fragmented material (2 g−1 and 0.11 cm3 g−1 for 3DOM LSMO to 48.9 m2 g−1 and 0.15 cm3 g−1 for 3D-hm LSMO (a 46% increase), respectively. The increase in surface area (∼30%) arising from fragmentation is anticipated to derive primarily from the newly exposed (001) facet in the 3D-hm. In contrast, the BET surface area of the 1DDN LSMO is 4.3 m2 g−1, which is more than 10 times lower than the 3D-hm LSMO surface area.XRD analysis indicates that the selected calcination conditions (750 °C for 4 h) are suitable to form a crystalline material possessing a cubic symmetry : 0.3874, nm) which is comparable to the ideal LSMO perovskite-type structure (JCPDS PDF No. 04-016-6114)6sotherms . The isomaterial 10. Fragmδ+ species with a higher level of oxidation have been reported to be more active for CH4 oxidation than both Mnδ+ species with a lower valence and metallic Mn0 (ref. 2 g−1) and the highest surface Mn4+/Mn3+ molar ratio (1.51), as shown in 2− (Olatt), adsorbed oxygen (Oads), hydroxyl and/or carbonate groups and adsorbed water species vary non-monotonically with the different LSMO morphologies. The Oads/Olatt molar ratio increases from 0.35 to 0.44 upon using the fragmentation protocol (2-temperature programmed desorption (TPD) result  as well as a shift in the Olatt peak to a lower temperature (730 °C). The result demonstrates that the fragmentation process provides an LSMO surface enriched in Oads species and improves the mobility of the Olatt species. No obvious low-temperature desorption peak is apparent for the 1DDN LSMO as it likely possesses fewer Oads species on the surface. A higher Oads presence can facilitate the adsorption and activation of O2 molecules and promote the performance of the catalyst for CH4 oxidation9XPS measurements were undertaken to identify any effect the fragmentation process may have had on surface atomic ratios and elemental state changes in the 3D-hm LSMO. The results are summarized in Mn0 ref. . The 3D-protocol . The res) result , which r4 oxidation activity data  and 3DOM (102 kJ mol−1) LSMO are higher than for the 3D-hm LSMO sample (67.3 kJ mol−1). Ea) has with the Oads/Olatt molar ratio and BET surface area of the LSMO samples. The catalytic performance decreases in the sequence of 3D-hm LSMO>3DOM LSMO>1DDN LSMO and coincides with increases in both the BET surface area and Oads/Olatt molar ratio of the materials. The higher surface area, the mesopore presence, the greater amount of Oads species and the improved Olatt, mobility exhibited by the 3D-hm LSMO combine to play important roles in the oxidation of CH4. The 3D-hm LSMO provides a higher specific reaction rate compared to the pristine 3DOM LSMO on the basis of both mass (2.0 times improvement) and surface area (1.4 times improvement), indicating that the increased surface area arising from fragmentation is not the sole factor enhancing the 3D-hm LSMO catalytic performance.From the CHity data , the 3D-0−420 °C , where tEa value lower than what has been previously reported for CH4 oxidation  and (110) surfaces of LSMO were used to represent the exposed facets of the 3D-hm structure (−2) is lower than for the (110) surface (1.92 J m−2). The numbers agree well with other calculation values obtained for LaMnO3 (ref. 4 molecules than the (110) surface  SrO-terminated layer. On the MnO2-terminated slabs, CH4 molecules are the most stable when they are adsorbed on a Mn site with a H atom being directed towards the surface (Eb=−0.25 eV) followed by adsorption on an O site (Eb=−0.18 eV)  surface are included in To further understand the elevated activity exhibited by the 3D-hm LSMO catalyst, DFT calculations were employed to reveal the origin of the reduced activation barrier for CHtructure . The surnO3 ref.  and impl4 compared to the (110) surface on the 3DOM LSMO. The findings highlight the potential for using structural disassembly as a means of synthesizing unique nanoscaled particles with versatile properties and opens exciting possibilities for attaining highly effective materials for catalytic systems.An effective fragmentation strategy to generate a mesostructured 3D network composing of hexapod-shaped multi-metal oxide crystalline nanoparticles has been successfully exploited. The study has demonstrated that the fragmentation protocol invokes new structural and chemical features within the 3D-hm LSMO perovskite which elevate its catalytic activity for methane oxidation. The improved features include a modified mesoporous structure, a richness of surface oxygen species, better lattice oxygen mobility and exposure of the (001) facet along the fractured faces. DFT calculations indicated that the freshly exposed (001) surfaces of the 3D-hm LSMO impart a lower cleavage energy for the first C−H bond in CH3)3.6H2O, Sr(NO3)2, Mn(NO3)2, 3.0 ml ethanol, 3 ml ethylene glycol (EG) and 7.0 ml deionized water while solvent B comprised 0.7 g L-lysine, 0.4 g citric acid and 2.0 ml deionized water. After complete dissolution of each of the components, the two solvents were mixed together and stirred for 4 h to give a transparent solution. An aliquot of 2.0 g PMMA was added to the precursor solution and stirred for 4 h, followed by filtration and drying at 100 °C for 12 h. A two-step calcination process was utilized: the obtained solid was first calcined N2 flow (90 ml per min) at a ramp of 1 °C per min from RT to 300 °C, where it was maintained for 3 h. The material was then cooled to RT and calcined in air (90 ml per min) from RT to 750 °C at 1 °C per min where it was held for 4 h. 1DDN LSMO was prepared by a citrate methodTwo solvents, namely solvent A and solvent B, were prepared separately: solvent A comprised desired amounts of La with 1 min resting intervals over a 2 h time-frame. During the fragmentation process, the suspension temperature was maintained below 25 °C by an ice bath. After sonication, the suspension was centrifuged for 10 min at 5,000 r.p.m. and 15 °C to isolate the supernatant which contained the LSMO hexapods and fragmented legs. The suspension was collected while the settled solids were redispersed, sonified and centrifuged again following the same procedure. As a final step, the collected supernatants were dried at 100 °C for 12 h to obtain the 3D-hm LSMO perovskites. The preparation details of PMMA and the comparison methods of 3D-hm LSMO are specified in 2 (30 ml per min) at 300 °C for 2 h and then in a N2 (30 ml per min) as it cooled to RT. The reactant gas mixture containing 5 vol% CH4+30 vol% O2+65 vol% N2 was then introduced to the reactor at a total flow of 42.8 ml per min, thus giving a gas hourly space velocity of ca. 50,000 ml (g h)−1. The concentrations of the reactants and products in the reactor effluent were monitored on-line by a gas chromatograph (Young Lin 6500) equipped with a thermal conductivity detector (TCD) detector and a Carboxen-1010 PLOT column. The catalytic activities of the samples were evaluated using the temperatures T10%, T50% and T90% required for methane conversions of 10%, 50% and 90%, respectively. CH4 conversion was determined using (cinlet−coutlet)/cinlet × 100%, where cinlet and coutlet are the CH4 concentrations in the inlet and outlet feed streams, respectively.Catalytic activity was assessed using a fixed-bed quartz tubular microreactor (i.d.=6.0 mm) at atmospheric pressure for the complete oxidation of methane, as shown in Scheme S3. The catalyst sample (50 mg) was first pre-treated in a pure O2-TPD analysis was on a Micromeritics Autochem II apparatus. XPS analyses were performed on a Thermo Scientific, UK (model ESCALAB250Xi). The operation conditions are listed in XRD analyses were performed on a PANalytical Empyrean II diffractometer. SEM, TEM and HAADF-STEM images were collected on FEI Nova NanoSEM 450 FE-SEM microscope, Philips CM200 apparatus and JEOL JEM-ARM200F STEM, respectively. The specific surface areas and pore size distributions were obtained on a Micromeritics Tristar 3030 adsorption analyser and O−5 eV tolerance, and forces were optimized until they were no more than 5 × 10−2 eV Å−1. K-points were sampled using 4 × 4 × 1 and 4 × 3 × 1 mesh. To improve the electronic description of LSMO used a Hubbard correction with DFT, as implemented in Vienna ab initio simulation package (VASP)The authors declare that all the other data supporting the findings of this study are available within the article and its How to cite this article: Wang, Y. et al. The controlled disassembly of mesostructured perovskites as an avenue to fabricating high performance nanohybrid catalysts. Nat. Commun.8, 15553 doi: 10.1038/ncomms15553 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Methods, Supplementary Discussion, Supplementary Tables and Supplementary References


Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes.Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family  The scaffolding protein eIF4G binds to both and thus links the 5′- and 3′-ends of the mRNA resulting in its circularization. In plants, eIF4E and eIF4G shape the eIF4F complex, whereas eIF4A is probably loosely attached to the complex and does not co-purify (The m7G(5′)ppp(5′)N cap-structure that is covalently linked to the 5′ end and the poly(A) tail at the 3′ end are critical o-purify , 2012. Ao-purify  suggesticis-acting elements and mechanisms (Translation initiation is rate limiting and a highly regulated step . While mchanisms . It is wTombusviridae and the genus Luteovirus have been shown to contain cap-independent translation enhancers in the 3′-end of their genome(3′-CITEs)  . Their m′-CITEs) . Their mterminus), for several 3′-CITEs it has been shown that the ribosome has to scan from the 5′-end of the RNA, as insertion of stable SL-structures at the 5′-terminus , able to enhance cap-independent translation of many animal virus and some eukaryotic mRNAs . But whiterminus  or inserterminus .Construction and analysis of chimeric viruses have demonstrated that 3′-CITEs can be exchanged between viruses, suggesting that they are functionally independent RNA elements. Since the publication of the last review on 3′-CITEs , a new cin vivo in protoplasts, since in some cases the 3′-CITEs are not functional in vitro in wheat germ extract (WGE).To date, seven different classes of 3′-CITEs have been identified. Their structures are very variable, therefore 3′-CITE presence is difficult to predict and identification has to be based on functional analysis. In most cases activity has been assayed by flanking a reporter gene (for example luciferase) with viral 5′- and/or 3′-end sequences and analyzing the translation efficiency of these constructs preferably Barley yellow dwarf virus (BYDV) is the best characterized and was named BYDV like-element (BTE) . It is present in all members of the genera Luteovirus (family Luteoviridae) and Necrovirus, Dianthovirus, and Umbravirus (family Tombusviridae) . The bases in lower case can vary, but the consensus GNRNA  in the apical loop is conserved (boxB RNA (The 3′-CITE of viridae) . The BTEviridae) . BTEs coonserved . This seboxB RNA . Wang etboxB RNA .Satellite tobacco necrosis virus (STNV), a parasitic subviral RNA that encodes only its capsid protein; it was named translation enhancer domain (TED) . The STNV TED was shown to be functional in enhancing translation in vitro and in vivo, being capable of functionally replacing a 5′-cap; also, it has been shown to confer cap-independent translation in vitro when moved to the 5′-untranslated region (UTR) of an uncapped reporter (Pelargonium line pattern virus (PLPV), the recommended type member of the new Pelarspovirus genus belonging to the family Tombusviridae, was shown to contain a TED-like 3′-CITE  was considerably larger (49 nt) than previously proposed , Elderberry latent virus (ELV), Rosa rugosa latent virus (RrLDV) and Pelargonium ringspot virus (PelRSV) , a member of the genus Carmovirus  its translation enhancer activity could be confirmed for genomic (g) and subgenomic (sg) RNAs in in vivo translation experiments, showing that it is dependent on the presence of the genomic and subgenomic 5′-ends in cis (The first 3′-CITE that was discovered was the one present in in (TED) . Its strin (TED) , but thireporter . Recentl 3′-CITE . In siliproposed . An enlaV genome . Apart f(PelRSV)  and addirmovirus . While is in cis .Panicum mosaic virus (PMV)  that was later shown to be similar in structure to the one identified in the umbravirus Pea enation mosaic virus RNA 2 (PEMV2) (Tombusviridae) . These 3′-CITEs were named PTE . PTEs are also found in several carmoviruses, as experimentally identified in Saguaro cactus virus (SCV) (Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), Honeysuckle ringspot virus (HnRSV), Pea stem necrosis virus (PSNV), Hibiscus chlorotic ringspot virus (HCRSV) and Galinsoga mosaic virus (GaMV). The other two known panicoviruses, Cocksfoot mild mosaic virus (CMMV) and Thin paspalum asymptomatic virus (TPAV), as well as one of the known aureusviruses, Pothos latent virus (PoLV), also seem to contain a PTE (viridae)    and propin a PTE . PTEs coin a PTE . Wang etin a PTE .Tombusviridae family: the tombusvirus Maize necrotic spot virus (MNeSV) and the carmovirus Melon necrotic spot virus (MNSV) . The ISS consists of a stem-loop structure of about 60 nt with a 5–8 nt final loop and two internal bulges protruding from the stem. The first structure of the MNeSV determined by SHAPE analysis (Maize white line mosaic virus (MWLMV) and Johnsongrass chlorotic stripe mosaic virus (JCSMV) and the tombusvirus Cucumber Bulgarian virus (CBV). Although there is considerable sequence divergence between the 3′-UTRs of all known MNSV isolates and MNSV-264, the 3′-CITE of MNSV-264 maintains the I-shape  and have been shown to be active in the 3′-UTRs of viruses belonging to different genera of the s (MNSV)   can be predicted to have 3′-CITEs with a conserved Y-shaped structure (YSS) (Tomato bushy stunt virus (TBSV) . Another experimentally demonstrated YSS 3′-CITE is the one from Carnation Italian ringspot virus  (Cymbidium ringspot virus (CymRSV), Cucumber necrosis virus (CNV), Artichoke mottled crinkle virus (AMCV), Pelargonium necrotic spot virus (PNSV), Grapevine Algerian latent virus (GALV), and Pelargonium leaf curl virus (PLCV) have been proposed to contain a YSS  , as shows (TBSV)   . The tomin a YSS . The thrin a YSS . Mutatioin a YSS , supportTurnip crinkle virus  . A similar structure was proposed for the related carmovirus Cardamine chlorotic fleck virus (CCFV). Additionally, apart from its PTE two functional TSSs have been identified in the PEMV2-3′-UTR, one upstream of the PTE, the kl-TSS, and the other near the 3′-end of the genomic RNA, the 3′-TSS, with both predicted to fold into structures similar to tRNAs . Although both are essential for virus accumulation in vivo, mutations that disrupted the TSS near to the 3′-end had no effect on translation   has a thviridae) . This isviridae)  . It was shown that this region is implicated in RdRp binding, inducing the conformational change of the TSS possibly by disruption of the H4a/ψ3 pseudoknot that leads to loss of the H4b/H5 interaction (The RNA-dependent RNA polymerase (RdRp) has been shown to bind the 3′-end of the TCV genome leading to a conformational change of the TSS region . The reseraction . This coFigure 3C). This translational enhancer was named CXTE, as it had been acquired by MNSV from an Asiatic isolate of Cucurbit aphid-borne yellows virus  , is also small (55 nt) and consists of two helices protruding from a central hub . Since iviridae) . Thus, MTombusviridae do not resemble any of the known 3′-CITE classes  and is sufficient to facilitate translation alone. On the other hand, eIF4E alone does not promote BTE-mediated translation, but the presence of both subunits of eIF4F results in a 25% increase in translation efficiency, while the affinity of BTE binding to eIF4F is four-fivefold higher (Kd = 37 nM)  . This co= 37 nM) . ThroughFigure 1 . AnalysiFigure 1 . Sharma Figure 1 . The autFigure 1 . The preFigure 1 .Kd of 30 and 50 nM, respectively  even in the absence of eIF4G, with the binding affinity to eIF4F being very similar .In contrast to the previous 3′-CITEs, the PTE of PEMV2 has been shown to bind eIF4E with high affinity  or mutation (G47C) strongly reduced its eIF4F binding capacity .The ISS of MNeSV has been shown to interact with wheat eIF4F in WGE . In thiscapacity . These Gin vitro binding assays showed that Ma5TE also binds eIF4F: eIF4Fp20, formed by purified recombinant melon eIF4E with a 20 kDa truncated eIF4G fragment, was shown to be competent for Ma5TE binding, with the Kd estimated to be 2 μM , which were paired and thus not accessible without eIF4F, became accessible. Mutations in these residues affected the in vivo cap-independent translation activity of Ma5TE, while no binding of eIF4Fp20 to Ma5TE A4109C could be observed in vitro . Interestingly, mutation of the corresponding adenosine residue in MNeSV 3′-CITE (reducing its flexibility by pairing it) also strongly affected the activity of this ISS .Mapping of the eIF4F binding sites in Ma5TE by footprinting analysis revealed that, in the presence of eIF4Fin vitro . These tthis ISS . This suFigure 2C), although it has been shown to be able to function in the absence of eIF4E. MNSV-264 is able to infect melon plants with reduced eIF4E expression due to its specific silencing, in contrast to MNSV-Mα5 that is unable to infect , with high homology to the 5′-end of the CABYV 3′-UTR, are still unknown. The CXTE is functional in melon plants silenced for eIF4E, thus it may be eIF4E-independent .Arabidopsis eIF4G-deficient mutant . In these cases, the 3′-CITE activity is 5′-end dependent and mutations disrupting base-pairing affect translation, while compensatory mutations restore activity. The interacting sequences are usually unpaired, thus located in conserved apical loops of hairpins .Figure 1). This BTE was functional even if complementarity was achieved by insertion of foreign sequences outside the BTE   . The pre origin) . Two alt origin) : either Red clover necrotic mosaic virus  , where no effect on translation was observed by mutating complementary sequences between BTE and 5′-UTR (The only exception is the BTE-like 3′-CITE (named 3′TE-DR1) of d 5′-UTR . In thisd 5′-UTR . The ARSd 5′-UTR .in cis . However, this could not be confirmed by mutational analysis, as disruption of this potential base-pairing reduced translation only slightly, while restoring complementarity by mutations at the opposite end did not restore translation . The complementary sequence in the 5′- end (involving 6–8 bases) is located in the viral p27 ORF . In the case of the carmovirus SCV the functionally important 5′ complementary sequence is located in the loop of a hairpin in the coding region of p26 . This interaction was shown not to interfere with eIF4F binding, while ribosome scanning was dependent on the ISS and occurred from the 5′-end . For the two aureusviruses MWLMV and JCSMV and the tombusvirus CBV as well, complementary sequence stretches between the 5′-end and the loop of their predicted ISSs could be identified. Thus, in the ISS-mediated translation activity, the eIF4F bound to the 3′-CITE reaches the 5′-end through 5′–3′ interaction.As with the other 3′-CITEs described above, 5′–3′-UTR interactions based on sequence complementarity have been identified between the apical loop of the only SL forming the ISS of MNeSV and the loop of the first SL of the 5′-UTR  and showe 5′-end . Similare 5′-end  and compFigure 2D). Moreover, the YSS of CIRV was also shown to require a 5′–3′ interaction between its YSS and 5′-UTR, based on sequence complementarity, in order to bring the eIF4F (or isoform) complex bound to the YSS to the 5′-end . Additionally, for both TBSV and CIRV YSS ribosome scanning has been shown to occur from the 5′-end . The hairpin at the 5′-end involved in this interaction is located in the coding region of the first ORF. This interaction was shown to be critical for virus translation and multiplication, and its loss affected translation to a greater extent than deletion of the adjacent PTE. This suggested that the 5′–3′ interaction supported additional translation functions besides bringing the PTE bound eIF4E to the 5′-end  which binds the 60S ribosomal subunit , 2013    , similar to the ISS. Toe-print analysis of the H4 4E-SE in the presence of eIF4E showed RT-stops at the A and the following C, suggesting that eIF4E binding involves this loop  ; in the gure 2C) ; and fingure 2C) . Interesgure 2C)  also havhis loop .Figures 2A,C (arrows), 4A]. Thus, these two residues could be involved in eIF4E binding in these 3′-CITEs. In the case of the PTE structures, these two residues are less conserved . Additionally, one or two guanosine residues can also be identified in ISS and TED CITEs upstream of the apical loop in bulge 2 in a similar position as the G-domain of PTEs, suggesting their possible role in eIF4E binding . Also, the paired G/C present in the final stem that became accessible in the presence of eIF4E in MNSV ISS can be identified in all TED and PTE CITEs as a G/C (or C/G) pair .Analysis of the residues present downstream the apical loop in bulge 1 of the assayed or predicted structures of TED 3′-CITEs allows for the identification, as in all the ISSs, of A and G residues (adjacent or separated by a maximum of 2 nt) conserved in all of them [Figures 4A,B). If 3D structural predictions of the left helix of the YSS of CIRV or TBSV are superimposed on the prediction of the ISS from MNeSV , it can be observed that the G downstream of the apical loop in bulge 2 and A and G in bulge 1 of MNeSV seem to be localized at similar positions in both YSSs. This prediction may suggest that binding of eIF4F to YSS also occurs through the eIF4E subunit as shown for the ISS, PTE and TED. This, however, has to be further studied.The five residues proposed above to play a role in eIF4F binding through eIF4E can also be identified in the YSSs, located in the left arm of the Y  and the BTE from Tobacco necrosis virus (TNV-D). While maintaining the sequence complementarity between the 3′-CITE and the 5′-UTR, these chimeric viruses were able to translate and multiply efficiently, with the exception of the BTE-substitution, suggesting that 3′-CITEs are functionally interchangeable elements.The diversity of 3′-CITEs in closely related viruses suggested that these RNA elements may be interchanged between different viruses. To test this hypothesis, Figure 5). This stretch of 55 nt was shown to act itself as a 3′-CITE in presence of the 5′-UTR in cis  and MNSV-USA-2012 and 2014, that have low sequence identity with MNSV-264, although their 3′-UTRs are highly similar . All these MNSV variants suggest that acquisition of 3′-CITEs through recombination might be frequent and/or positively selected for.From an evolutionary perspective, the absence of correlation between virus taxonomy and the presence of different classes of 3′-CITEs  suggestsR in cis . The traR in cis . InteresNicotiana benthamiana, a non-host for MNSV, thanks to its new 3′-CITE ; it is possible that this sequence also acts as a 3′-CITE in CABYV. Thus, some classes of 3′-CITEs appear in viruses of different genera or even families, although others are quite conserved within virus genera, like the YSSs that have been identified only in tombusviruses, or BTEs which have been identified in phylogenetically related viruses (3′-CITES are modular, functional RNA elements that can be exchanged between viruses, and their acquisition may contribute to a gain of function resulting in a selective advantage, broadening the virus host range. Interestingly, in MNSV-N, the CXTE has been acquired by interfamilial recombination with CABYV, a virus belonging to a different family ( viruses . This maVT wrote review. MM and MA edited and added specific information to all sections.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.


Influenza A virus is a pathogen of global medical importance causing significant health and socio-economic costs every year. Influenza virus is an unusual pathogen in that it is pleomorphic, capable of forming virions ranging in shape from spherical to filamentous. Despite decades of research on the influenza virus, much remains unknown about the formation of filamentous influenza viruses and their role in the viral replication cycle. Here, we discuss what is known about influenza virus assembly and budding, focusing on the viral and host factors that are involved in the determination of viral morphology. Whilst the biological function of the filamentous morphology remains unknown, recent results suggest a role in facilitating viral spread in vivo. We discuss these results and speculate on the consequences of viral morphology during influenza virus infection of the human respiratory tract. Between 1918 and 1920, an estimated 50–100 million people died from the first recorded pandemic of a viral infection commonly known as the flu .IAV is a pleomorphic virus of the Orthomyxoviridae family consisting of a segmented, single stranded, negative sense RNA genome . ResponsIAV infects the cells of the upper respiratory tract, causing illness in a wide range of hosts, including humans, pigs, horses and birds. Birds are thought to be the reservoir for IAV in the wild  and are The IAV genome comprises of 11 genes on eight RNA segments. These can broadly be categorised as encoding the viral structural or non-structural proteins. Haemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP) and matrix proteins one and two (M1 and M2) are primarily structural proteins with additional functional roles, whilst the polymerase subunits  and the non-structural proteins (NS1 and NS2) serve mainly function roles during virus replication. NP binds to and is involved in the packaging of the viral genome along with the polymerase subunits for transport and assembly . HA is rIt is thought that IAVassembly and budding occurs at lipid raft domains on the apical surface of the host cell plasma membrane, where IAV proteins are brought together in high concentrations within specific membrane regions . NA and IAV is a pleomorphic virus, known to display a range of morphological states, from filamentous to spherical, with ovoid or bacilliform intermediates often observed . In certMany different studies have investigated the viral factors that determine morphology, with most focusing on the structural proteins M1 and M2 and their role in viral assembly. M1 plays a crucial role in the assembly and budding of both filamentous and spherical IAV , 24. In Interestingly, in filamentous viruses, M1 appears to adopt a helical conformation , which iIAV is pleomorphic, adopting both spherical and filamentous forms . As descIn 2010, Bruce et al. reported that Rab11 and Rab11-family interacting protein 3 (FIP3) are necessary for the formation of filamentous virus . Rab11 iThe biological significance of IAV morphology in human clinical infections is a subject of great interest. The production of viral filaments appears to be highly inefficient by its nature, consuming anywhere from three to thirty times the amount of plasma membrane used to bud one infectious virus , 53. TheIn 1998, it was shown that spherical and filamentous viruses are comparably infectious in vitro and both contain a single copy of the viral genome , 69–71. In this review, we have explored recent findings in how IAV is assembled and budded and how some of these mechanisms, especially from a cellular aspect, may influence the budding of filamentous IAV. Whilst many studies have found an effect of cellular proteins and viral protein sequences on filament formation, the causation and function of filamentous IAV remains to be fully understood. What recent data shows for certain is that IAV morphology is not solely cell or virus dependent but relies on a precise balance and interaction between the two. A single point mutation in a viral protein can alter morphology whereas silencing of a single cellular gene can also affect the ability of an IAV strain to form filamentous virions. Furthermore, a strain that forms filamentous virions in vivo will not necessarily do so in vitro or in a different host species. Ultimately, the role of filamentous IAV in human infection remains an enigma. Given the resource requirements needed to produce a single filamentous IAV as compared to a single spherical virion, it is likely that the filamentous morphology confers some advantage to the virus. Future investigations of IAV host-pathogen interactions will undoubtedly shed more light on the mechanisms of viral morphogenesis, allowing for a better understanding of the mechanisms of influenza virus budding, the functions of viral morphology and the impact of morphology on influenza disease in human clinical infections.


Tibrovirus currently has three official members assigned to two species: Bivens Arm virus and Tibrogargan virus (species Tibrogargan tibrovirus) and Coastal Plains virus . Here, we report the complete genome sequence of a new putative member of this genus, Beatrice Hill virus. Although relatively closely related to the three classified viruses, Beatrice Hill virus represents a novel Tibrovirus species.The rhabdoviral genus  Rhabdoviridae currently includes 13 genera and four unassigned species  and Tibrogargan virus (TIBV) have been assigned to the species Tibrogargan tibrovirus, and Coastal Plains virus (CPV) has been assigned to the species Coastal Plains tibrovirus , and CPV from an apparently healthy steer (–Culicoides insignis)  Ekpy steer –, and (c)ectively . All tibectively , 10.Culicoides peregrinus) that had been collected at Beatrice Hill, Northern Territory, Australia (Tibrovirus clade (k-mer length of 25 (Beatrice Hill virus (BHV) was first reported in 1984 as a novel virus of biting midges  based on its divergence from TIBV and BAV.Using PASC , the detKY073493.The GenBank accession number of Beatrice Hill virus isolate CSIRO 25 is


Cryptococcus neoformans and C. gattii,acquired from the environment. In Latin America, as occurringworldwide, C. neoformans causes more than 90% of the cases ofcryptococcosis, affecting predominantly patients with HIV, while C.gattii generally affects otherwise healthy individuals. In thisregion, cryptococcal meningitis is the most common presentation, withamphotericin B and fluconazole being the antifungal drugs of choice. Aviandroppings are the predominant environmental reservoir of C.neoformans, while C. gattii is associated withseveral arboreal species. Importantly, C. gattii has a highprevalence in Latin America and has been proposed to be the likely origin ofsome C. gattii populations in North America. Thus, in therecent years, significant progress has been made with the study of the basicbiology and laboratory identification of cryptococcal strains, in understandingtheir ecology, population genetics, host-pathogen interactions, and the clinicalepidemiology of this important mycosis in Latin America.Cryptococcosis is a life-threatening fungal infection caused by the encapsulatedyeasts  Cryptococcus neoformans,and C. gattii. Topics related to epidemiology, clinical data andtreatment, molecular studies on clinical and environmental isolates, host-pathogeninteractions, laboratory data on antifungal susceptibility, preliminary data detectingantigenemia in patients, and studies to search for the environmental niche of the fungusare presented here.In Latin America, the study of cryptococcosis and its etiological agents has becomeincreasingly important, as this mycosis has significant morbidity and mortality, withmore than 5,000 individuals affected with cryptococcal meningitis each year, and 2,400attributable annual deaths in Latin America alone. The main goal of this review is topresent recent and relevant data regarding the studies done in Latin American countrieson cryptococcosis and the etiological agents Epidemiology - C. neoformans is the most importantcause of fungal meningitis in the world, and in sub-Saharan Africa, meningealcryptococcosis is the most common type of meningitis in adults infected with HIV  inpatients with HIV, cryptococcosis is the second most common, after toxoplasmosis, whichhas been demonstrated in clinical studies ,and autMost cases of cryptococcosis are reported in young HIV positive male patients. Among theHIV negative patients, males are also the predominant sex, although in a smallerproportion, and patients also tend, on average, to be slightly older . This epClinical aspects - Cryptococcosis mainly affects the CNS, causingmeningitis. In a lesser proportion it affects the lungs, and organs such as skin, eyes,prostate, and bone, among others ,consists of a combination of amphotericin B deoxycholate (AmBd) 0.7 mg/kg/day, and5-fluorocytosine (5FC) 100 mg/kg/day for two weeks as induction phase, followed byfluconazole (FCZ) 400 mg daily for eight weeks as consolidation phase, and FCZ 200 mgdaily as the maintenance phase. In the induction phase it is recommended to use, as analternative, the use of liposomal AmB (LAmB) associated (or in combination) with 5FC fingerprinting  for C. neoformans var.neoformans serotype D isolates, VNIII (AFLP3) for hybrids betweenthe serotypes A and D, and VGI (AFLP4), VGII (AFLP6), VGIII (AFLP5) and VGIV (AFLP7) forC. gattii serotype B, and C isolates.printing , amplifiprinting , microsaprinting ,restricprinting , multiloprinting , and morprinting , have alCryptococcus and cryptococcosisreported from Latin America, the isolates have been identified only at species level,variety, or serotype; it is now possible to determine the major molecular type of thecryptococcal isolates recovered not only from clinical, but also from environmentalsources, and much less frequently from veterinary samples, by using one or more of theabove mentioned molecular techniques.Although in most of the studies on Using these techniques, the major molecular type of 3,486 isolates in Latin America ispresently known, with most of the isolates reported from Brazil (45.87%), followed byColombia (24.99%), Mexico (9.21%), Argentina (8.09%), Cuba (6.05%), Venezuela (2.90%),Peru (1.06%), Ecuador (0.77%), Chile (0.55%), Guatemala (0.43%), as well as Honduras,Paraguay, and Uruguay with one isolate each . From BoC. neoformans var.grubii, serotype A is the predominant molecular type (72.8%)causing cryptococcosis in Latin America, followed by C. gattiimolecular type VGII (AFLP6) (14.4%)   have been reported from veterinary cases:one molecular type VGI from Cuba , corresponding to  (14.4%) .Interesii VGII) ,althougisolates . Of all rom Cuba  and fiverom Cuba  Fig. 1)C. neofor testing. To dateC. gattii has been isolated as etiological agent,have been documented much more recently , bronchial lavage (BL), biopsy,and, cerebrospinal fluid (CSF), showing a sensitivity and specificity of 100%  the bases of the preference of some genotypes for certain hosts (i.e.immunosuppressed vs. immunocompetent) and target organs (i.e. brain vs. lung); (3) themechanism by which the infection becomes persistent and/or recurrent despite thatin vitro resistance to antifungal drugs does not seem to be aproblem for cryptococcal treatment; and (4) the diverse pathways related to sexualreproduction, among many others .orldwide  has stimin vitro andin vivo models.In Latin American, specifically in Argentina, Brazil and Cuba, different investigationsrelated with these topics have been conducted mostly in order to reveal and comprehendhost-pathogen interactions, to investigate the role of interactions between the yeastsand other microorganisms, and to review alternative approaches to antifungal treatmentsagainst cryptococcosis and other mycosis. A clear majority of the authors have attemptedthen to understand the role of the immune cells and the cytokines/antibodies inductionnot only from human clinical samples but also by using in vitro stimulation with heat-killed C.gattii and C. neoformans (40 different strains),demonstrated that clinical heat-killed C. gattii isolates induced amore pronounced inflammatory response with higher concentrations of pro-inflammatorycytokines  compared toother Cryptococcus species and non-clinical C. gattii,which is dependent on TLR4 and TLR9 as cellular receptors  antigens, priming naïve andre-stimulating infected animals to induce T-cell and B-cell responses, and thedevelopment of a Th1 microenvironment with increased levels of nitric oxide (NO)synthesis and C. neoformans-specific immunoglobulin production, whichall lead to a protective immune response against subsequent infection with fungus. Alsoin rats, the mechanisms involved in macrophage apoptosis promoted by cryptococcalglucuronoxylomannan (GXM) through NO generation showed to be novel immunomodulatorymechanisms that could contribute to limit inflammation during infection in these groupof rodents (C. neoformans (strain 028 LMIPK) by macrophages without fungicidaleffect, thus favouring yeast dissemination and decreasing the survival of mice due tocryptococcal infection  or H. capsulatum alone, or acapsulated strains, as shownby higher pulmonary fungal burden in co-infected animals. Enhanced pellicle formationwith a hybrid polysaccharide matrix with higher reactivity to GXM mAbs and increasedresistance to phagocytosis and killing by macrophages was also observed, which togetherindicate that a microbial interaction involving the transfer of virulence traits maytranslate into enhanced lethality during mixed fungal infections (C. gattii (strain L27/01) was demonstrated.Germ-free (GF) mice were more susceptible to infection, showing lower survival, higherfungal burden in the lungs and brain, increased behavioural changes, reduced levels ofinflammatory cytokines , and lower NFκBp65 phosphorylationcompared to conventional mice, which were associated with smaller yeast cells andpolysaccharide capsules in the lungs, and less tissue damage. Moreover, macrophages fromGF mice showed reduced ability to engulf, produce reactive oxygen species (ROS), andkill C. gattii. Restoration of microbiota in mice that received fecesfrom conventional animals or administration with Escherichia coli, mademice more responsive to infection, which was associated with increased survival andhigher levels of inflammatory mediators (C. gattiiinfection (strain L27/01) and the concurrent infection with influenza A virus (IAV)strain H1N1 was studied. This co-infection resulted in a major increase in morbidity andmortality, with severe lung damage and a high brain fungal burden when mice wereinfected in the acute phase of influenza multiplication, indicating that IAV infectionis a predisposing factor for severe disease and adverse outcomes in mice co-infectedwith C. gattii. IAV not only alters the host response to the yeast,leading to recruitment of significantly more neutrophils and macrophages into the lungs,but also induces the production of type 1 interferons (IFN-α4/β) and significantlyreduces the levels of IFN-γ, which is associated with an impaired immune response.Reduced phagocytosis, killing of cryptococci and production of ROS by IAV-infectedmacrophages were also shown, leading to increased proliferation of the fungus withinmacrophages  in C. neoformans H99infection was also revealed. Gal-3 levels no only augments in human sera but also inspleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are moresusceptible to cryptococcosis than wild-type animals, as the first group present higherfungal burden and lower animal survival. In vitro experiments showedthat Gal-3 also inhibits fungal growth and exerts a direct lytic effect on C.neoformans EVs, which could benefit the host (Both the host .C. neoformans strain 102/85 (HKC)emulsified in complete Freund adjuvant (CFA), promoted protection against anintraperitoneal challenge with viable C. neoformans var.grubii with significantly better clearance of yeasts from tissues,a lower concentration of GXM in serum and tissues, and better histopathologicalparameters compared to un-pretreated infected rats. Passive immunisation with a mAb toglucosylceramides (GlcCer), a lipid that is present at the fungal plasma membrane, cellwall, and secretory vesicles, and which induces antifungal antibodies and regulates thevirulence of C. neoformans in animal infections, significantly reducedhost inflammation and prolonged the survival of mice lethally infected with C.neoformans strain 24067 (Serotype D), revealing a potential therapeuticstrategy to control cryptococcosis , linked to the effector Fc regionof murine IgG2a (WGA-Fc), was able to inhibit in vitro fungal growth,to increase phagocytosis by macrophages, as well as their antifungal functions , the Clinical Laboratory Standard Institute (CLSI) microdilution method fromM27-A3, and the EDef 7.2 from the European Committee on Antimicrobial SusceptibilityTesting (EUCAST). Several additional techniques have been developed to simplify thestandard ones, such as, time kill curves (TKC), E-Test strips, and a disk diffusionmethod  and FC, showing thatFC allowed to determine MIC for AmB in a 2 h incubation time, with MICs data obtained onthe same day  were determined. ECVs are commonly used to separatewild-type (WT) isolates from isolates with reduced susceptibility to antifungal drugswith probable acquired resistance mechanisms, are useful to monitor the emergence ofstrains with mutations that could lead to reduced antifungal susceptibility toantifungal drugs, however they should not be used as predictor of clinical outcome. Inthe study by In three additional studies, the epidemiological cut-off values (ECV) for AmB, 5FC, FCZ,itraconazole (ITZ), posaconazole (PCZ) and voriconazole (VCZ), were established  and receClinical and environmental data - The studies are presented in C. gattii strains are more resistantto FCZ in comparison to clinical C. neoformans strains. Otherwise, forAmB and 5FC no difference has been reported. Another general observation is thatenvironmental isolates are more susceptible to antifungal drugs than clinicalisolates.As a general observation, clinical C. neoformans and C. gattii. Clinical studies arewarranted to ascertain the relevance of this occurrence as a tool for guide treatment.Additionally, resistance to FCZ was studied by C. neoformansclinical isolates. The sequencing revealed the G1855A mutation in three isolates,resulting in the enzyme amino acid substitution G484S. These strains were isolated fromtwo fluconazole-treated patients. Importantly, this mutation would not intervene in thesusceptibility to the other widely use azoles, ITZ and VCZ.It is important to mention the work by de Novel compounds for treatment - The synthesis of novel compounds hasbeen explored by one research group in Colombia  as the natural habitat of C. gattii, serotype C,VGIII , an enzyme that oxidizes diphenolic substrates to form long polymersof melanin. Laccase provides protection to the fungus in the environment, and it is oneof the most important and most studied virulence factors inCryptococcus  is the medium of choice forenvironmental studies. These seeds provide the substrate for the laccase and the endproduct is melanin, which is associated with the cell walls and gives the colonies acharacteristic brown pigment allowing for the differentiation from other yeastcolonies.Many questions about the presence of the fungus in the environment were still without acomplete answer, and one of them was related to the survival time of the blastoconidiain the samples. In this way, a couple of studies were carried out in Latin America, andthe findings indicated that blastoconidia could remain viable from 45 days up to tenyears . EffortsC. gattii, the pioneer environmental studies that have beendone in Latin America and the ongoing ones with the assistance of molecular tools willallow to understand more clearly the expansion of the ecological niche of thisspecies.Related to C. gattii in British Columbia,Canada  globally, C. neoformans moleculartype VNI, causes more than 90% of the cases of cryptococcosis in Latin America and themain risk factor for acquiring it (70-90%) is to be infected with HIV. C.gattii, VGII and VGIII, are also recovered, affecting otherwise healthyindividuals; (ii) AmB and FCZ are the antifungal drugs of choice, since 5FC isunavailable in the region; (iii) laboratory diagnoses and species or molecular typeidentification are performed with the standardised protocols; (iv) determination of theantifungal susceptibility shows that in 90% of cases, both species are susceptible tothe antifungals tested, namely: AmB, 5FC, FCZ, PCZ, VCZ, and ITZ. Particularly,C. gattii VGIII, serotype C isolates have shown decreasedsusceptibility to azoles, especially to FCZ; (v) molecular studies of clinical andenvironmental isolates show that in Latin America, C. neoformans,molecular type VNI, is not only predominant among the clinical cases, but also in theenvironment, followed by C. gattii molecular type VGII, with similarproportions in both clinical-, and environmental sources; (vi) a great amount ofinformation is from the Latin American studies on the environmental niche, especiallyfor C. gattii; (vii) the increasing application of genotyping methodsin Latin America, has also greatly contributed to the global study of C.neoformans and C. gattii, to the recognition of theirgreat inter-, and intra-species genetic diversity, to understand how these pathogenicyeasts have spread around the world and what is their population structure, and tobetter define some disease aspects of cryptococcosis, an important mycosis in theregion.


This paper describes a new computer virus spreading model which takes into account the possibility of a virus outbreak on a network with limited anti-virus ability. Then, the model is investigated for the existence of equilibria and their stabilities are proved and illustrated. Moreover, it is found that these two factors are not only relative to the threshold value determining whether the virus becomes extinct or not, but that they are also relative to the virus epidemic levels. Theoretical and experimental results indicate that, in some ways, it would be practically possible to eradicate the virus or suppress its prevalence below a suitable level. Consequently, some suggestions are proposed that may help eradicate or suppress virus propagation over a real computer network. SIR model , once infected, any susceptible computer is in its latency , and secondly, that by using the anti-virus software on the affected network, the virus can be completely and immediately eliminated. However, these assumptions are inconsistent to the facts that, in general, most viruses break out suddenly. Zuo et al , due to a possible connection with infected computers, every susceptible computer is either latent with probability (1-p) βI(t)dt, or breaks out with probability pβI(t)dt, where p>0 is a constant. Since the existing anti-virus ability of any given network is limited, the virus is temporarily suppressed with probability ε, where ε > 0 is a constant. At time t, every infected computer is either latent sequentially with probability ε or recovered with probability α.Let From the work above, the transmission between the model can be expressed by the following system of differential equations:R, we can consider the following reduced model:Because the first three equations in (1) are independent of S(0) ≥ 0, E(0) ≥ 0, I(0) ≥ 0 and all the parameters are positive constants. It is easily verified that the setWith initial conditions R0 for the model and it is defined as the number of virus-free computers that are infected by a single infected computer. It is easy to see that the model (2) always admits a virus-free equilibrium E0 = . Then letThis section is devoted to understanding the dynamic behaviors of the model (2). First, we will obtain a threshold value Then,Hence, next generation matrix for the model isR0 of the matrix K is the basic reproduction number of the model, i.e.Again, the spectral radius E0 isModel (2) always has a unique equilibrium μ and other roots are decided by the following equations:Clearly, the eigenvalues are −R0 < 1, then a = μ + γ − pβS0 + ε + α + μ > 0, andIf The two roots of Eq  both havR0 < 1, the virus-free equilibrium E0 is local stability.When Now, it is turn to examine the global stability of virus-free equilibrium by use of the Lyapunov direct method. The following Theorem is obtained:R0 ≤ 1, the virus-free equilibrium E0 is global stability.When Proof. DefineR0 < 1 with I = 0 or R0 = 1. Hence, V is a lyapunov function on Ω. Thus, I → 0 as t → ∞. I = 0 in the first equation of model (2) shows that S → b / μ as t → ∞. Therefore, it follows from the Lasalle’s invariance principle that every solution of the model, under the initial conditions in Ω, approaches E0 as t → ∞.As all the model parameters are positive, it follows that Example 1. Consider model (2) with parameters b = 5, β = 0.07, μ = 0.6, γ = 0.25, ε = 0.3, p = 0.7 and R0≈0.57<1. The condition of virus-free equilibrium stability is satisfied and hence the virus-free equilibrium E0 is asymptotically stable . By calculations, the unique equilibrium And the corresponding characteristic equation of the linearization of model (2) isa1a2–a3 > 0 holds, then according to the Routh-Hurwitz criterion, the following theorem is given:It is easy to verify that E* is locally asymptotically stable when R0 > 1.Virus equilibrium Remark 1. This Theorem expresses that, under the assumption of virus outbreak on a network with limited anti-virus ability, virus epidemic would attain a certain level, and the model admits no the periodic and chaotic dynamic behaviors.E* is globally asymptotically stable.Virus equilibrium Proof. We consider the following Lyapunov function constructed asMeanwhile, for simplicity, denoteV(t) along the solution of Eq (on of Eq  isV(t)'By using the following equationsEq  can be wx > 0 and the equality holds if and only if x = 1; y,z > 0 and the equality holds if and only if y = z; x,y,z > 0 and the equality holds if and only if x = y = z. Therefore, V(t)' ≤ 0 for x,y,z > 0 and V(t)' = 0 holds if and only if x = 1 and y = z. Thus, it follows from LaSalle Invariance Principle that the virus equilibrium E* is globally asymptotically stable.Since the arithmetical mean is great than or equal to the geometrical mean, thus, Example 2. Consider model (2) with parameters b = 5, β = 0.15, μ = 0.6, ε = 0.03, p = 0.9, γ = 0.4 where R0 = 1.90>1 satisfies the stable conditions for the virus equilibrium , and hence the virus equilibrium is asymptotically stable  with parameters b = 5, β = 0.15, μ = 0.6, p = 0.5, γ = 0.5, the global dynamical behavior of every variables of the model (2) with varying parameterεis shown in Fig 3ε-S(t), Fig 4ε-E(t) and Fig 5ε-I(t).Example 4. Consider model (2) with parameters b = 5, β = 0.15, μ = 0.6, ε = 0.2, γ = 0.5, the global dynamical behavior of every variables of the model (2) with varying parameter p is shown in p-S(t), p-E(t) and p-I(t).Theorem 3.2 and 3.3 tell us, to some degree, it would be possible to eradicate the virus or inhibit its epidemics below a proper level in practice. In order to do this, it is critical to have an overall knowledge of the effects of parameters on the virus propagation. By the proof of Theorem 3.1, we havep and transmission rateε play a key role, then differentiating R0 with respect to them, we can obtain:Since breakout probability Hence, we obtain the following Theorem:Theorem 4.1 Consider model (2), the following claims hold trueR0 is increasing with p, and is decreasing with ε.Example 5. Consider the effect ofε, p on R0 in model (2) with parameters b = 5, β = 0.075, μ = 0.6, γ = 0.5, R0 is increasing with p and decreasing withε.I*. A direct calculation yields:Next, we are ready to study the effect of parameters on virus epidemic level Theorem 4.2 Consider model (2), the following claims hold trueI* is increasing with p and decreasing with ε.Example 6. Consider the effect ofε, p on I* in model (2) with parameters b = 5, β = 0.15, μ = 0.6, γ = 0.5, I* is increasing with p and decreasing with ε, respectively.Based on analyses above, the following suggestions may help eliminate or suppress the spread of computer viruses in a real network:R0 is well below one, Theorem 4.2 demonstrates that R0 is more sensitive to change in p than inε. This sensitivity informs us that outbreak is more important than passive detection for computer viruses, which leads us to eliminate the virus by dissecting its logical structure [When tructure , analysiR0 is significantly greater than one, it leads us to suppress virus propagation to an acceptable level .When By taking into consideration the possibility of a virus outbreak on a network with limited anti-virus ability, a new computer virus spreading model is proposed and analyzed. The results are as follows:R0, which determines whether the virus may become extinct or not. More specifically, R0 is proportional to the former and inversely proportional to the latter.The outbreak probability and transmission rate are strongly relative to the threshold value I*. More specifically, I* increases with the former and decreases with the latter.epidemic levelsThe outbreak probability and transmission rate are heavily connected with the virus epidemic level R0 ≤ 1 whereas the virus equilibrium is globally stable if R0 > 1.The virus-free equilibrium is globally stable if These results indicate that, in some ways, it would be practically possible to eradicate the virus or suppress its prevalence below an acceptable level. Theoretical and experimental results reveal the effects of the parameters on virus propagation. Consequently, the proposed suggestions may help eradicate or suppress virus propagation over a real computer network.The main goal of this paper is to better understand the propagation of computer virus qualitatively rather qualitatively. In our view, our work give an insight into the investigation of computer virus spreading by incorporating the objective realistic factors into compartment modeling, which could attract growing attention and interest. Meanwhile, the proposed model is based on the homogeneous network, which can be expanded into heterogeneously node-based model in complex networks, and it is one of our next objectives.


Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222  and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored. Orthomyxoviridae. Migratory waterfowl are the natural reservoirs for IAVs, but these viruses also infect humans, domestic poultry, wild birds, pigs, dogs, cats, horses, mink and marine mammals, including seals and whales1 Human IAVs bind preferentially to N-acetylneuraminic acid-α2, 6-linked galactose  receptors, whereas avian influenza viruses (AIVs) prefer N-acetylneuraminic acid-α2,3-linked galactose  receptors.4 Swine are considered a ‘mixing vessel' of IAVs because they have both Neu5Acα2, 3-Gal and Neu5Acα2,6-Gal receptors throughout their respiratory tract. With these receptors, swine facilitate the generation of novel influenza reassortants and enable avian-like IAVs to obtain the ability to bind to human receptors,5 as has been hypothesized to have occurred during the genesis of viruses that caused the 1957 H2N2 and 1968 H3N2 influenza pandemics.6Influenza A virus (IAV) is an enveloped, segmented, single- and negative-stranded RNA virus belonging to the family 7 Most H6 viruses introduced from waterfowl into domestic poultry have gained only limited spread. However, during 2000–2005, subtype H6N2 IAVs caused illness outbreaks among domestic poultry in CA, USA.10 In addition, H6 IAVs have been shown to replicate well in mice without pre-adaptation, indicating that these viruses could cause cross-species infection in mammals.12 Laboratory experiments showed that humans can be infected with H6 IAVs through experimental inoculation.13 Furthermore, findings from serologic surveillance suggested that veterinarians exposed to H6 IAV-infected domestic birds can become infected with the virus,14 and in 2013, an avian-origin H6N1 IAV was reported to cause human infection, but there has been no evidence of subsequent human-to-human transmission.15H6-subtype IAVs have been detected in various migratory waterfowl and domestic poultry in Eurasia and North America.18 and some of these H6 viruses recognized human receptors.19 In 2010, after an avian-origin H6N6 swine influenza A virus (SIV) was isolated from sick pigs in southern China, it was found that the virus had been transmitted to and was circulating among the swine population; seroprevalence rates ranged from 1.8% to 3.4%.21 The hemagglutinin (HA) protein of the currently circulating H6N6 SIV has amino acids 222V and 228S, compared with amino acids 222A and 228G in its potential AIV precursors. In other IAV subtypes, HA amino acids 222V and 228S have been reported to affect virus replication in mammals.22Since 2002, H6 IAVs have been one of the predominant IAV subtypes circulating in live bird markets in southern China,23 After its discovery in humans, this virus quickly moved to swine and other animal populations worldwide.28 During the past few years in southern China, A(H1N1)pdm09 virus has become one of the predominant viruses among domestic swine.31 Frequent reassortments between influenza A(H1N1)pdm09 and other endemic SIVs have been observed.33The virus that caused the 2009 H1N1 pandemic, influenza A(H1N1)pdm09, was a swine-origin IAV.The aims of our study were to understand the impacts of two acquired mutations in HA of H6N6 virus on its receptor-binding properties and to assess the transmission potential of the H6N6 virus. We also aimed to assess the potential risks posed by reassortants of H6N6 virus with A(H1N1)pdm09 virus because such reassortants would be expected to emerge if both viruses continue to circulate in swine.20 To study the virus, we used an RNeasy Mini Kit  to extract RNA from the isolate; extraction was performed in a Biosafety Level 3 (BSL-3) laboratory.In 2010, an avian-like H6N6 SIV, A/swine/Guangdong/k6/2010(H6N6), was isolated from swine in southern China.2.Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293 T cells  were used for virus propagation; the cells were maintained in Dulbecco's Modified Eagle Medium . A549 cells (American Type Culture Collection) used in assays were maintained in Advanced DMEM/F-12 (Gibco/BRL). The medium for each of the three cell lines was supplemented with 10% fetal bovine serum , penicillin–streptomycin, and amphotericin B (Gibco/BRL), and the cells were held at 37 °C in 5% CO34 The site-directed mutagenesis on residues 222 and 228 of HA was performed using a QuikChange II Site-Directed Mutagenesis Kit . Primers are available upon request.The full-length cDNA for eight genes of A/swine/Guangdong/k6/2010(H6N6) virus were amplified by using the SuperScript One-Step RT-PCR system  and then cloned into a pHW2000 vector.35 The recombinant viruses were confirmed by Sanger sequencing at the Life Sciences Core Laboratories Center at Cornell University .Eleven recombinant viruses were generated  by rever35Hemagglutination and hemagglutination inhibition (HI) assays were carried out by using 0.5% turkey erythrocytes as previously described.N-linked glycans, Manα1,6-Manβ1,4-GlcNAcβ1,4-GlcNAc (N32) and Neu5Acα2,3-Galβ1,4-GlcNAcβ1, 2-Manα1,6-Manβ1,4-GlcNAcβ1,4-GlcNAc (N52) were synthesized to represent as N-acetylneuraminic acid-α2,3-linked galactose  and Manα1,6-Manβ1,4-GlcNAcβ1,4-GlcNAc (N33) and Neu5Acα2,6-Galβ1,4-GlcNAcβ1,2-Manα1,6-Manβ1,4-GlcNAcβ1,4-GlcNAc (N53) were synthesized to represent as N-acetylneuraminic acid-α2,6-linked galactose . The N-linked glycans were first labeled by 2-amino-N-(2-amino-ethyl)-benzamide (AEAB) as described previously36 and then biotinlyated by using EZ-Link NHS-LC-LC-Biotin  according to the manufacturer's instruction. Glycan quantities were measured by high-performance liquid chromatography .The biotinlyated α2,3- and α2,6-sialic acid receptors  were purchased from GlycoTech . N-linked glycans, 3′SLN and 6′SLN) and the viruses in a PBS solution (pH=7.4) containing 0.01% bovine serum albumin and 0.002% Tween-20  with 10 μM neuraminidase inhibitor  and 10 μM oseltamivir phosphate . The binding assay was performed by using a FortéBIO Octet K2 interferometer equipped with streptavidin biosensor tips (FortéBIO Inc.). In summary, the biotinylated receptors were first coated onto the biosensor tips for 300 s, after which the tips were dipped into a 1 mg/mL protein concentration of virus for 1200 s (association step) and then into the kinetics buffer with neuraminidase inhibitors for 1000 s (dissociation step). The entire measurement cycle was maintained at 30 °C with orbital shaking at 1000 × rpm.The glycan stock solution (1 mg/mL) was prepared in 50% glycerol in 1 × phosphate-buffered saline (PBS) (v/v), according to the manufacturer's instructions. The protein concentration in viruses was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. For the binding analysis, we further diluted the sialic acid receptors  or Opti-MEM I containing TPCK-treated trypsin. At 12, 24, 48 and 72 h after inoculation, supernatant (200 μL) was collected from the cells and titrated, by 50% tissue culture infectious dose (TCID50), in MDCK cells.To determine the growth kinetics, we inoculated the MDCK and A549 cells with rgH6N6-222V/228S, rgH6N6 × pdm09-222V/228S, rgH6N6-222A/228G, rgH6N6 × pdm09-222A/228G, rgH3N2 × pdm09 , wild-ty2 for 1 h. The inocula were then aspirated, and the cells were overlaid with 2 mL of 1% agarose containing TPCK-treated trypsin (1.5 μg/mL). Cultures were incubated for 3 days at 37 °C and then fixed with methanol and stained with 1% crystal violet to reveal plaques.Plaque assays were performed on MDCK cells in six-well tissue culture plates. Serial dilutions were prepared from the virus stock, and 800 μL of each dilution was incubated in MDCK cells at 37 °C with 5% CO37 The virus labeling, glycan microarray hybridization and data analyses were performed as previously described.38The viruses were purified using 25% sucrose as previously described.6 TCID50 viral load in a 1-mL volume) and the other as an exposure ferret to be exposed to the virus through indirect  or direct contact with the virus-inoculated ferret. Before the experiments were conducted, all 24 ferrets tested negative for antibodies to rgH6N6-222V/228S (wild-type-like), A/California/04/2009(H1N1), A/Perth/16/2009(H3N2), A/Victoria/361/2011(H3N2) and A/Minnesota/307875/2012(H3N2) influenza viruses. In the aerosol transmission groups, the virus-inoculated and exposure ferrets were housed in the same cage on different sides of a 1-cm-thick, double-layered, steel partition with 5-mm perforations . The airflow in the cage went from the exposure ferret to the virus-inoculated ferret. In the direct-contact transmission groups, the virus-inoculated and exposure ferrets were housed together in the same cage without a partition. In all cages, the exposure ferret was put into the cage 1 day after the virus-inoculated ferret was inoculated with a testing virus.To test the transmissibility of the two testing viruses (rgH6N6-222V/228S and rgH6N6 × pdm09-222V/228S), we designed six experiment groups for each virus: three aerosol transmission and three direct contact transmission groups. Two 4-month-old female ferrets (Triple F Farms) were included in each of the 12 groups: one as a virus-inoculated ferret to be inoculated intranasally with a testing virus  and from exposure ferrets at 1 DPI; thereafter, nasal wash fluids were collected every other day until 10 DPI for both groups of ferrets. Body temperature and weight were measured before nasal wash fluids were collected.50 in MDCK cells and confirmed by 50% egg infectious dose (EID50) in 9-day-old embryonated chicken eggs.Serum samples were collected from all ferrets at 14 DPI, immediately before they were killed. Virus titers in nasal wash fluids were determined by TCID50 in MDCK cells.To test the replication efficiency of testing viruses in the ferret respiratory track, we killed two of the three virus-inoculated ferrets in each contact transmission group at 3 DPI. The turbinates, trachea, bronchi and lungs were collected, and virus titers were determined by TCIDAll laboratory and animal experiments were conducted under BSL-2 conditions, with investigators wearing appropriate protective equipment, and in compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Mississippi State University.39 We used GARLI version 0.9640 and maximum likelihood criteria to perform phylogenetic analyses, and we conducted bootstrap resampling analyses with 1000 runs by using PAUP* 4.0 Beta41 with a neighbor-joining method as described elsewhere.42We conducted multiple sequence alignments by using the MUSCLE software package.Phylogenetic analyses showed that the HA genes from H6N6 SIVs and H6 AIVs, including the strain that caused human infection, belong to different sublineages within a Eurasian lineage . The PB2To investigate whether substitutions A222V and G228S (avian to swine) affect virus-binding affinity to erythrocytes, we conducted hemagglutination assays with eight reassortant viruses generated by reverse genetics: rgH6N6 × PR8-222V/228S, rgH6N6 × PR8-222A/228S, rgH6N6 × PR8-222V/228G, rgH6N6 × PR8-222A/228G, rgH6N6 × pdm09-222V/228S, rgH6N6 × pdm09-222A/228S, rgH6N6 × pdm09-222V/228G, and rgH6N6 × pdm09-222A/228G . ErythroN-linked glycans . Results showed that the rgH6N6-222V/228S (wild-type-like) virus can bind to Neu5Acα2,3-Gal  and Neu5Acα2,6-Gal , but the affinities to Neu5Acα2,3-Gal  were at least two-fold higher than those to Neu5Acα2,6-Gal  on the receptor-binding properties of H6N6 virus, we also performed binding assays using a FortéBIO system with a set of Neu5Acα2,3-Gal and Neu5Acα2,6-Gal glycans: two glycan analogs (3′SLN and 6′SLN) and four synthetic The glycan array, which contained a total of 152 α2,3-linked and α2,6-linked glycans, was used to determine the receptor-binding profile of H6N6 SIV and the effect of mutations V222A and S228G on the glycan-binding profile of H6N6 IAV. Results showed that rgH6N6 × PR8-222V/228G and rgH6N6 × PR8-222A/228G had binding affinities below the detection threshold for the majority of the α2,3-linked and α2,6-linked glycans on the glycan array . However3.67 to 103.83 TCID50/mL, and at 2 DPI, titers peaked at 104.5 TCID50/mL; viral shedding continued until 5 DPI in these ferrets (We used a ferret model to determine the transmissibility of rgH6N6-222V/228S virus by direct and indirect (aerosol) contact. In the direct-contact transmission experiment, the rgH6N6-222V/228S virus-inoculated ferrets did not show obvious clinical signs of illness. At 1 DPI, nasal wash fluids from these ferrets had virus titers ranging from 10 ferrets . HI assa ferrets . Ferrets ferrets .3.00 to 104.00 TCID50/mL, and virus titers peaked at 2 DPI at 104.50 TCID50/mL; these virus-inoculated ferrets continued to shed viruses until 6 DPI , supporting that H6N6 virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets.4.67–106.00 TCID50/mL; at 2 DPI, virus titers peaked at 106.33 TCID50/mL and continued to shed until 6 DPI pdm09 virus, we determined transmissibility of the reassortant virus by direct contact and aerosol contact in ferrets. The three rgH6N6 × pdm09-222S/228V virus-inoculated ferrets had weight loss and slightly elevated body temperatures at 3 DPI, but the ferrets showed clinical recovery from 4 DPI onward. Nasal wash fluids collected from the virus-inoculated ferrets at 1 DPI had virus titers of 10il 6 DPI . All thril 6 DPI .4.00–104.50 TCID50/mL in nasal wash fluids at 4 DPE  enabled influenza A(H1N1)pdm09 viruses to acquire dual receptor specificity for complex α2,3-linked and α2,6-linked sialic acids; the substitution also increased the virulence of this virus.47 We recently showed that the W222L substitution in HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinity of the virus to canine-specific receptors with Neu5Acα2,3-Galβ1,4-(Fucα-) or Neu5Acα2,3-Galβ1,3-(Fucα-)-like structures.38 In addition, mutation W222R in HA can increase influenza virus infectivity in mice49 by introducing a hydrogen bond between the virus HA and the host glycan receptor.50Compared with its progenitor AIV, H6N6 SIV has mutations 222V and 228S in the 220-loop of its HA. Mutations in this 220-loop  have been shown to affect receptor-binding specificity. For example, mutations Q226L and G228S enabled H3 viruses to bind sialic acid α2,3-Gal and sialic acid α2,6-Gal receptors.51 and that guinea pig erythrocytes disproportionately express α2,3- and α2,6-linked sialic acids.52 The receptor-binding profiles we obtained in this study with turkey, chicken, guinea pig, dog and horse erythrocytes suggest that mutations V222A and G228S, which are associated with a change in affinity from avian to swine, especially mutation G228S, changed receptor-binding specificity. This mutation G228S led to a minimum 16-fold increase of receptor-binding affinities for guinea pig and horse erythrocytes and a minimum two-fold decrease in binding affinities to chicken erythrocytes pdm09 virus did not increase transmissibility of H6N6 SIVs through aerosol or direct contact. Nonetheless, it is possible that additional mutations acquired through adaptation or genetic reassortments between H6N6 SIV and influenza A(H1N1)pdm09 virus would increase the transmissibility of H6N6 SIV.A number of studies have shown that AIVs, such as highly pathogenic H5N1 and low pathogenicity H9N2 viruses, have increased aerosol transmissibility after acquiring the internal genes of influenza A(H1N1)pdm09 virus.In the pathogenesis studies, virus replicated in lungs of rgH6N6 virus–infected ferrets, but the ferrets exhibited no obvious signs of illness. Virus titers in lungs of rgH6N6 × pdm09 virus-infected ferrets were ≥5-fold higher than those in rgH6N6 virus-infected ferrets. In addition, ferrets infected with rgH6N6 × pdm09 virus showed slightly elevated temperatures and weight loss at 1 and 2 DPI.We showed that the method used for detecting virus loads in nasal wash fluids or tissues affects the accuracy of the results. For example, embryonated chicken eggs were more sensitive than MDCK cells in detecting H6N6 SIVs in the virus transmission group: viruses in aerosol-exposed ferrets were detectable in embryonated chicken eggs but not in MDCK cells .In summary, our findings suggest that subtype H6N6 virus can bind to α2,6-linked sialic acids, indicating H6N6 virus as a virus with zoonotic potential. Although H6N6 SIV has limited transmissibility between ferrets and probably cannot yet be transmitted between ferrets through infectious aerosolized droplets, the virus could evolve into a more transmissible H6 virus through additional adaptation and reassortment. Thus, evolution of this H6N6 virus and other H6 AIVs should be closely monitored.


Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. A 1358-nt region of the NS5 gene was amplified and sequenced but an isolate was not recovered.We previously reported the discovery of a novel, putative flavivirus designated T’Ho virus in The complete genome of T’Ho virus was sequenced using a combination of unbiased high-throughput sequencing, 5′ and 3′ rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. The genome contains a single open reading frame of 10,284 nt which is flanked by 5′ and 3′ untranslated regions of 97 and 556-nt, respectively. Genome sequence alignments revealed that T’Ho virus is most closely related to Rocio virus (67.4% nucleotide identity) and Ilheus virus (65.9%), both of which belong to the Ntaya group, followed by other Ntaya group viruses (58.8–63.3%) and Japanese encephalitis group viruses (62.0–63.7%). Phylogenetic inference is in agreement with these findings.This study furthers our understanding of flavivirus genetics, phylogeny and diagnostics. Because the two closest known relatives of T’Ho virus are human pathogens, T’Ho virus could be an unrecognized cause of human disease. It is therefore important that future studies investigate the public health significance of this virus. Flavivirus (family Flaviviridae) contains more than 70 viruses, most of which are transmitted to vertebrates by arthropod vectors such as mosquitoes and ticks  tails were added to the 3′ ends of the T’Ho virus genomic RNA using 6 units of poly (A) polymerase . Tailing reactions were performed at 37 °C for 1 h and then terminated by heat-inactivation (65 °C for 10 min). Poly (A)-tailed RNA was reverse transcribed using a poly (A) tail-specific primer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′). Complementary DNAs were PCR amplified using a forward primer specific to the T’Ho virus cDNA sequence and a reverse primer that matched the 5′ half of the poly (A)-specific reverse transcription primer (5′-GGCCACGCGTCGACTAGTAC-3′). PCR products generated from the 5′ and 3′ RACE reactions were inserted into the pCR4-TOPO cloning vector  and ligated plasmids were transformed into competent TOPO10 http://www.ebi.ac.uk/Tools/msa/clustalo/) were performed to calculate percent nucleotide and amino acid identities between select sequences.The genomic and predicted amino acid sequences of T’Ho virus were aligned to all other sequences in the Genbank database by application of BLASTn and BLASTp, respectively . Sequenc6 generations with 25% burn-in, resulting in maximum PSRF 1.005 among all continuous parameters) [The complete ORF sequences of T’Ho virus and other flaviviruses were aligned using Muscle v3.8.31 . Phylogeameters) , neighboameters)  and maxiameters) . The GTRameters) .The T’Ho virus genome consists of 10,937 nt (Genbank Accession No. EU879061.2) and contains a single 10,284-nt ORF flanked by 5′ and 3′ UTRs of 97 and 556-nt, respectively. The ORF encodes the three structural and seven nonstructural proteins common to all known flaviviruses. The position and length of each gene and untranslated region are shown in Table Cleavage sites in the T’Ho virus polyprotein are shown in Table http://www.cbs.dtu.dk/services/NetNGlyc/) with the consensus sequence defined as Asn-X-Ser/Thr (where X is not Pro) in the context of specific surrounding sequences. Fourteen Asn-X-Ser/Thr motifs were identified in the T’Ho virus polyprotein sequence. Eight motifs are predicted to be utilized by N-linked glycans: these are located at prM15, prM148, E154, NS1130, NS1207, NS2A122, NS5234 and NS5655.Potential N-linked glycosylation sites were identified using the NetNGlyc 1.0 Server  listed in the Ninth Report of the International Committee on Taxonomy of Viruses Fig. . Cell fuCx. quinquefasciatus from the Yucatan Peninsula of Mexico. The closest known relatives of T’Ho virus are ROCV and ILHV, that both belong to the Ntaya group of mosquito-borne flaviviruses, with 67.4% and 65.9% nt. identity, respectively. Based on genetic criteria, we propose that T’Ho virus is classified as a new species within the genus Flavivirus. It has been suggested that flaviviruses with >84% nucleotide sequence identity should be classified within the same species [We report the complete genome sequence of T’Ho virus, a novel putative flavivirus discovered in  species . The gen species ; howeverThe two closest known relatives of T’Ho virus are recognized human pathogens. ROCV was responsible for several epidemics of severe encephalitis in Brazil in the 1970s , 17, 44.Aedes and Psorophora spp. mosquitoes (particularly Ps. ferox) with birds implicated as principal reservoir hosts [Culex spp. mosquitoes and birds [Aedes spp. mosquitoes and primates [Culex spp. mosquitoes and birds [Culex, Aedes and/or Psorophora spp. mosquitoes and the principal reservoir hosts are birds.An isolate of T’Ho virus is not available; thus, experimental infection studies cannot currently be performed to assess the vector and reservoir competence of mosquito and vertebrate species likely to be involved in its amplification. However, probable vectors and reservoir hosts of T’Ho virus can be inferred from the information available for its closest known relatives. The amplification cycles of ROCV and ILHV are not well defined but most isolations have been made from ir hosts , 31, 34.nd birds , 22, 43,primates . JE ground birds . It is tFlavivirus, whose closest known relatives are human pathogens; thus, it is feasible to suggest that T’Ho virus may be an unrecognized cause of human disease in the Yucatan Peninsula of Mexico. Our report enables directly the creation of specific PCR diagnostic assays. However, serological cross-reactivity is common with flaviviruses [In conclusion, we describe a novel species, T’Ho virus, of the genus iviruses , as exemiviruses . Accordi


Introduction: Grenada is a rabies endemic country, where terrestrial rabies is maintained in the small Indian mongoose (Herpestes auropunctatus). The role of bats in the epidemiology of rabies in Grenada is unknown. A 1974 report described one rabies virus positive Jamaican fruit bat (Artibeus jamaicensis), and a high seroprevalence in this species. In the current study, the natural exposure to rabies virus in Grenadian bats was re-evaluated. It is postulated that bats serve as a natural rabies reservoir, probably circulating a bat-specific rabies virus variant.Material and methods: Bats were trapped in 2015 in all six parishes of Grenada using mist- and hand nets. For the detection of rabies virus in brain tissue, the direct fluorescent antibody test (dFAT) and the reverse transcription polymerase chain reaction (RT-PCR) were used. Serum neutralizing antibodies were determined using the fluorescent antibody virus neutralization test (FAVN).Results and discussion: Brain tissue and sera from 111 insectivorous and frugivorous bats belonging to four species were tested . Rabies virus antigen and genomic RNA were not detected in brain tissues. Rabies virus neutralizing antibodies were detected in the sera of eight A. jamaicensis in four of the six parishes. Bats in Grenada continue to show natural exposure to rabies virus. As rabies virus was not isolated in this study, serology alone is not sufficient to determine the strain of rabies virus circulating in A. jamaicensis bats in Grenada.Conclusion: Artibeus jamaicensis appears to play a role as a reservoir bat species, which is of public health concern in Grenada. Dispersion of bats to neighboring islands is possible and serological bat surveys should be initiated in these neighboring states, especially in those areas that are free of rabies in terrestrial mammals. Lyssavirus genus that can cause rabies, rabies virus (RABV) is the only one that circulates in the New World.[Rabies lyssavirus (RABLV) infection is enzootic in bats in North America, where the virus has been detected in most known bat species. Of the 1ew World. Many RABew World. However,ew World.Herpestes auropuntatus) with spillover into domestic animals. The RABV variant that circulates in Grenadian terrestrial mammals has recently been described.[Artibeus jamaicensis).[A. jamaicensis.In Latin America and the Caribbean, rabies surveys and control measures have focused on terrestrial RABV reservoirs, and on the hematophagous or vampire bat species where they exist. Information on other bat species as potential rabies reservoirs in the Neotropics is scarce. This is also true for Grenada, one of four Caribbean islands where rabies is endemic, and is maintained in the small Indian mongoose (escribed. The roleicensis). The sameIn the current study, the natural exposure to RABV in Grenadian bats was re-evaluated. The working hypothesis is that Grenadian bats serve as a natural rabies reservoir, with circulation of a bat-specific variant of rabies virus. Implications for the spread of bat rabies virus to neighboring islands and the public health consequences are discussed.St George’s University (Grenada) Institutional Animal Care and Use Committee (IACUC) approved all animal work on 3 December 2014 under IACUC # 14008.Artibeus jamaicensis, Artibeus lituratus and Glossophaga longirostris were collected during the day using hand nets at their roosting sites  and at night using mist nets in forested and urban areas. Because of Molossus molossus’ flight behavior (high flight), mist nets were placed in front of their roosts to capture emerging bats at dusk. Bats were placed in individual cloth bags, transported to the lab and euthanized by isoflurane overdose. All bats looked healthy at the time of collection. Blood samples were taken immediately after euthanasia via cardiac puncture. Sera were heat inactivated for 30 min at 56°C and stored at −20°C until processing. Brains were removed and stored dry at −20°C until processing.TM Rabies DFA reagent, Millipore, Livingston, UK) was employed following supplier’s instructions.Rabies was diagnosed by the detection of viral antigen in acetone fixed brain tissue using the direct fluorescent antibody test (dFAT). A cocktaTM Biospec Products, Bartlesville, OK, USA) and using RNeasy Mini Kit spin columns  following the manufacturer’s instructions. RABV RNA was detected via RT-PCR as recently described.[Total RNA was extracted from 20 to 30 mg brain tissue after tissue lysis in a bead-beater  and may the presence of rabies virus neutralizing antibodies was measured in heat inactivated sera using the fluorescent antibody virus neutralization (FAVN) test with a fixed quantity of rabies virus  as previously described. Titers aConfidence limits at 95% for the reported seroprevalence rates were calculated using MedCalc statistical software .A. jamaicensis, two A. lituratus, 33 M. molossus, and 24 G. longirostris. Their trapping locations are shown in Figure 1, and bat species and numbers per parish are shown in A total of 111 bats belonging to four species were examined: 52 A. jamaicensis ; three were females and five males. Seropositive bats were trapped in four of Grenada’s six parishes  when non-flying infants were excluded. We showed that 15.4% (8/52) of A. jamaicensis were seropositive, all of which were flying juveniles or adults. There were no seropositive animals in the other three species trapped in our study, which may be due to an insufficient sample size: in the previous study,[M. molossus and G. longirostris was below 2%, and A. lituratus had not been captured.In Grenada, terrestrial and bat rabies co-exist. The results from our study for  of 1977. These auus study, the seroMyotis myotis), which ‘occurred with periodic oscillations in the number of susceptible, immune and infected bats’.[A. jamaicensis bats from Grenada indicates that these factors may explain differences in seroprevalence rates in the same species of bats from different years. It could be assumed that if the population size decreases below a minimum threshold that the virus would be expected to fade out. These seroprevalence data, however, suggest that the virus is being maintained within A. jamaicensis bats in Grenada and that the virus endemically circulates at a low and fluctuating level. The question of how infection is maintained in the absence of significant observed disease or mortality needs to be resolved to fully understand the public health implications.The differences in seroprevalence rates between the previous and the current study are not surprising. A 12-year longitudinal study has demonstrated significant inter-annual variations and cyclic lyssavirus infection in greater mouse-eared bats , continues to show natural exposure to RABV. There was no detection of RABV in the brains of our bats, indicating that they did not suffer from clinical rabies at the time of capture. This is not surprising, as all bats were trapped during flight or while roosting, which indicates that they were probably healthy at the time of sampling. So far, RABV is the only member of the Lyssavirus genus that has been described in the New World.[Lyssavirus genus within phylogroup I,[Our study confirms that at least one bat species in Grenada, the Jamaican fruit bat  fell below detectable levels 140 days after infection.[RABV is believed to have evolved in bats with subsequent spill-over into terrestrial mammals. Many repnfection. The actunfection. On the onfection. It must nfection. Other honfection.,22Myotis lucifugus) were inoculated intramuscularly, mimicking bite exposure. More than half of the infected bats succumbed to rabies, but their saliva was not infectious.[A. jamaicensis appears to form the largest colonies, with several hundred individuals. This might explain why the current and the previous study in Grenada [While seroprevalence rates reported elsewhere are generally high, relatively few rabid bats are reported and RABV detection rates in bat population studies are typically around or below 1% depending on sampling criteria. There isfectious. Followinfectious. In colonfectious. In Grena Grenada  observedIt is unknown whether transmission from Grenadian bats to terrestrial mammals or humans has ever occurred. A recent study describing the RABV variant that circulates in the Grenadian small Indian mongoose with spillover into domestic animals has shown that a monophyletic clade exists, distinct from the usual bat-associated variants found elsewhere. This doeA. jamaicensis populations is not monophyletic within islands. This indicates that these bats disperse among islands, perhaps facilitated by heavy storms and hurricanes.[The consistent finding of natural exposure to RABV in bats in Grenada remains a public health concern. Anyone exposed to a bat in Grenada, especially by being bitten, should seek immediate medical assistance. In most circumstances, especially when the bat is not available for testing, a full course of post-exposure prophylaxis should be administered to the patient as a precaution and following WHO guidelines. This is rricanes.In summary, Grenadian bats are exposed to rabies lyssavirus (RABLV) and may constitute a natural rabies virus reservoir, posing a potential threat to other mammalian species in Grenada and neighboring islands. It would be prudent to initiate active surveillance through serological bat surveys in neighboring states, even if these are considered rabies-free. Passive and disease surveillance studies are underway to determine the rabies lyssavirus variant circulating in Grenadian bats. These include testing clinically sick or dead bats, which is likely to be more successful in isolating the virus.