Peribunyaviridae family are naturally occurring reassortants, a common phenomenon for segmented viruses. Using a minigenome-reporter and virus-like particle (VLP) production assay, we have accessed the potential of Oropouche virus (OROV), Schmallenberg virus (SBV), and other orthobunyaviruses within the Simbu serogroup to reassort. We found that the untranslated region (UTR) in the medium segment is a potential contributing factor for reassortment by the tested viruses. We demonstrate that for promoter activity to occur it was essential that the viral RNA polymerase (L) and nucleocapsid (N) proteins were from the same virus, reinforcing the hypothesis that the large and small segments that encode these proteins segregate together during genome reassortment. Our results indicate that, given the right epidemiological setting, reassortment between SBV and OROV would potentially be feasible and could contribute to the emergence of a new Simbu virus.A number of viruses within the  Orthobunyavirus genus in the Peribunyaviridae family  comprises at least 30 viruses that can cause disease in humans and animals, including Oropouche virus (OROV), Schmallenberg virus (SBV) and La Crosse virus (LACV) [Orthobunyavirus genome is tripartite, made up of single-stranded segments of negative-sense polarity. The small genome segment encodes the nucleocapsid (N) protein and, in a majority of viruses, an additional nonstructural protein called NSs from an overlapping reading frame. The medium segment encodes a protein precursor for two envelope glycoproteins (Gn and Gc) and a nonstructural protein called NSm. The large genome segment encodes the RNA-dependent RNA polymerase (L protein). The 5′ and 3′ ends of these genome segments are flanked by highly conserved and complementary genus-specific sequences, which contain signals for genome transcription, replication, and packaging. Each genomic segment in combination with the N and L proteins form a functional ribonucleoprotein (RNP) [The s (LACV) ,2. The Oin (RNP) ,5,6.The segmented nature of the bunyavirus genome potentially allows for easy reassortment between its different members, a fact that was eloquently proposed a few years ago . This phOrthobunyavirus genus, could reassort, we utilised a minigenome and virus-like particle assay to test their protein-protein and protein-RNA interactions. OROV and SBV are both midge-borne and are classified in the Simbu serogroup. Whilst OROV causes a febrile illness in humans in South America [To investigate whether OROV and SBV, both important pathogens in the  America ,16, SBV  America ,18. Sepa2.BSR-T7/5 cells, which stably express T7 RNA polymerase  were groRenilla luciferase coding sequence as in [Renilla luciferase in the negative sense. Oligonucleotides containing the sequences corresponding to either the 5′ UTR or 3′ UTR of OYAV or Perdoes virus were then cloned in with excision PCR  and In-Fusion HD Cloning  techniques. This approach of synthetically cloning in the UTR sequences for OYAV and Perdoes virus was adopted due to the unavailability of viral stocks or plasmids for either virus. Oligonucleotides were based on sequences in GenBank.Minigenome-expressing and protein-expressing plasmids for OROV, SBV, and Bunyamwera virus (BUNV) have been previously described ,20,21,224 BSR-T7/5 cells/mL in 24-well trays were transfected with the desired amount of protein-expressing plasmids, 125 ng of a minigenome-expressing plasmid and 25 ng pTM1-FF-Luc using Lipofectamine 2000 , as per the manufacturer’s instructions. The amount of DNA in each well was kept constant by the addition of empty vector, pTM1. To normalize transfection efficiencies, cells were co-transfected with firefly luciferase expression plasmid pTM1-FF-luc, allowing induction levels of Renilla luciferase to be calculated [5 × 10lculated . At 24 h5 BSR-T7/5 cells/mL in 12-well trays were transfected with 500 ng of pTM1-L and pTM1-N, 125 ng of a minigenome-expressing plasmid, and 25 ng pTM1-FF-Luc. At 24 h p.t., Renilla and firefly luciferase activities were measured using Dual-Luciferase Reporter Assay kit (Promega). To generate virus-like particles (VLPs), the minigenome transfection mix was supplemented with 200 ng pTM1-M. At 48 h p.t., supernatants were harvested, clarified by centrifugation (1400 g for 5 min), digested with benzonase , and used to infect either BSR-T7/5 cells that were pre-transfected with 1 μg of pTM1-L, pTM1-N and pTM1-M for 24 h, or untransfected BHK-21 cells. Renilla activity was measured after 24 h using the Dual-Luciferase Reporter Assay kit (Promega).1.5 × 10To assess whether the N and L proteins of SBV recognise and initiate transcription and replication of the OROV minigenome, and vice versa, we compared minigenome activity with a combination of homologous and/or heterologous N and L proteins. To do this, we transfected BSR-T7/5 cells with OROV or SBV medium segment minigenomes (M-minigenome) with either the N and L protein-expressing plasmids of the respective virus or a combination of OROV and SBV N and L A.Renilla luciferase reporter gene flanked by viral UTRs, in the viral genomic (negative) sense, hence functioning as a genome segment analogue. Replication and transcription of this minigenome is initiated only when the N and L proteins can recognise the viral UTR as a functional promoter. This then leads to the expression of Renilla luciferase. As expected, minigenome activity was detected in the homologous minigenome systems  (The minigenome plasmids contain the SBV N-L) A. IntereSBV N-L) A. FurtheSBV N-L) A.Since SBV N and L could recognise and support OROV M-minigenome transcription and replication, we next tested their ability to utilise OROV small and large segment UTRs via S- and L-minigenomes. As shown in Renilla luciferase was detected from cells infected with OROV and SBV VLP controls  . Here, Bcontrols B, bar 6.controls B, which controls .As the interaction between viral Gn cytoplasmic tail (CT) and N or RNP is crucial for bunyavirus assembly , the resSince SBV N and L proteins were able to recognise OROV M UTR and not the other way around we decided to investigate the sequence difference that may contribute to this UTR recognition. We aligned the medium segment UTRs of OROV and SBV, along with that of other Simbu serogroup viruses—Akabane virus (AKV), Oya virus (OYAV), Perdoes virus, and BUNV A. IntereA minigenome assay with the plasmid bearing this mutation (OROV M-minigenome 8A/A 9A/A) resulted in a loss of UTR functionality with OROV N and L proteins and a decreased efficiency of UTR functionality with SBV N and L proteins  for AKV, OYAV, and Perdoes virus (in a previous publication we describe Perdoes virus as a reassortant containing OROV S and L segments ). The acOrthobunyavirus genus and vaccines based on attenuated viruses carry the risk of reverting back to pathogenicity due to genetic exchange with naturally circulating wild-type strains. Hence, understanding factors that drive reassortment will allow such vaccines to be implemented effectively.Segmented viruses carry the potential for genome reassortment, an important characteristic as this allows these viruses to generate new strains and new virus species. This phenomenon, on occasions, can result in the acquisition of new or increased pathogenic characteristics and, hence, understanding molecular determinants driving reassortment can be beneficial for predicting the emergence of a new virus. Additionally, the information gathered by studying reassortment may be applied to vaccine development. There are currently no vaccines available for any member of the Renilla luciferase activity was detected in both the OROV and SBV medium segment based minigenome systems , demonstrating the specificity of virus replication , the Gn CT acts as a matrix protein, bridging the RNA and glycoproteins for RNP trafficking and virus assembly [Peribunyaviridae family).Using minigenome systems, the current study focuses on SBV and OROV, two important Simbu serogroup viruses of veterinary and public health importance. As expected, lication A. Unexpe the RNP ,25, hencassembly . HoweverThe difference between the medium segment UTR nucleotide positions eight and nine of the clade A Simbu viruses  and the clade B Simbu viruses  is intriguing and it is tempting to speculate that these two residues may play a part in determining reassortment. The availability of complete orthobunyavirus sequences would be beneficial to allow a comprehensive analysis of this region and to determine if there is any relevance towards the virus–host pairing. For now at least, our results show that the N and L proteins of SBV exhibit a much broader UTR recognition range as it successfully transcribed and replicated AKV, BUN, OROV, OYAV and Perdoes virus M-minigenomes, unlike OROV or BUNV N and L ; Table 1Co-infection with bunyaviruses could theoretically result in eight potential genome combinations: AAA, ABA, BAA, AAB, BBA, ABB, BAB and BBB  . HoweverCulicoides midges, which have a wide geographic distribution [Both OROV and SBV are transmitted by ribution . The sprribution ,35. Take


Technical improvements in detection and enumeration of marine viruses  is an example of a ubiquitous virus in sea water, which causes muscle inflammation in Atlantic salmon. In this special issue, Haatveit et al.  provide 2 levels can affect the composition and diversity of Emiliania huxleyi viruses (EhV). Maat et al. )Prymnesium parvum, [Shewanella, [Vibrio anguillarum [Dinoroseobacter shibae [Shewanella, which forms biofilms in the gut of the tunicate Ciona intestinalis. Shewanella is part of a complex relationship between the C. intestinalis and its associated microbiome, and the study demonstrates that phage interaction with its Shewanella host contributes to the symbiotic relationships between the gut microbiome and the tunicate host  and bacwanella, , Vibrio uillarum  and Dinor shibae ), addingr shibae . The papr shibae  shows thate host .Synechococcus sp strain WH8102 during cyanophage infection suggested that the phage exploited the host genes for improved infection efficiency.Viruses may also acquire accessory genes from their eukaryotic or prokaryotic hosts  Figure . By exprMicromonas pusilla was reported by Cottrell and Suttle [Vibrio anguillarum are globally distributed either as free phages or as prophages inside bacterial genomes. The authors argue that selection for co-existence, rather than arms race dynamics, might explain the global distribution of near-identical H20-like bacteriophages and their prevalence as prophages in Vibrio genomes.The high local viral diversity obtained from oceanic metagenomic data  suggestsd Suttle , suggestd Suttle , which dEmiliania huxleyi, where the detection of viral RNA in the virus-resistant haploid cell of E. huxelyi suggested a new mechanism of infection, and the co-existence of viruses and host. Defense strategies are often associated with a fitness cost, as surface modification mutations may have an influence on, e.g., substrate uptake or enzyme secretion [Ostreococcus tauri [E. huxleyi [Viral host cells have developed multiple defense strategies against lytic viral infections . These iecretion , and bececretion . Such trus tauri , and E.  huxleyi . SurprisMicromonas and Ostreococcus [Prymnesium parvum [Emiliania huxleyi [The large and diverse group of nucleocytoplasmic large DNA viruses (NCLDV) includes a number of viral families infecting small photosynthetic protists, thus affecting mortality, evolution and production of these phytoplankton. In the current special issue, the research on NCLDV infecting phytoplankton is represented by the Prasinoviruses infecting the ubiquitous group of pico-sized Prasinophycea such as eococcus ,39,41, am parvum ,26 and E huxleyi ,31,36,40Acanthamoeba polyphaga mimivirus stimulated a new line of research, exploring the ecology and evolution of the group of large DNA viruses infecting eukaryotic protists, including the haptophyte Phaeocystis globosa [The discovery of the giant  globosa . Here, W globosa  synthesi3 of seawater, produce half the half of the oxygen in the atmosphere, and are integral to all known life. In a time where life in the oceans is under increasing threat , it is pressing to understand how viruses affect host population dynamics, biodiversity, biogeochemical cycling and ecosystem efficiency.The compilation of papers included in the current special issue highlights the exploration of eukaryotic and prokaryotic viruses, from discovery to complex interplays between virus and host and the interactions with ecologically relevant environmental variables. The discovery of novel viruses and new mechanisms underlying virus distribution and diversity exemplify the fascinating world of marine viruses. The oceans greatly shape Earth’s climate, hold 1.37 billion km


The Triassic Period saw the first appearance of numerous amniote lineages  that defined Mesozoic ecosystems following the end Permian Mass Extinction, as well as the first major morphological diversification of crown-group reptiles. Unfortunately, much of our understanding of this event comes from the record of large-bodied reptiles . Here we present a new species of drepanosaurid  from the Upper Triassic Chinle Formation of New Mexico. Using reconstructions of micro-computed tomography data, we reveal the three-dimensional skull osteology of this clade for the first time. The skull presents many archaic anatomical traits unknown in Triassic crown-group reptiles , whereas other traits  resemble derived avian theropods. A phylogenetic analysis of Permo-Triassic diapsids supports the hypothesis that drepanosaurs are an archaic lineage that originated in the Permian, far removed from crown-group Reptilia. The phylogenetic position of drepanosaurids indicates the presence of archaic Permian clades among Triassic small reptile assemblages and that morphological convergence produced a remarkably bird-like skull nearly 100 Myr before one is known to have emerged in Theropoda. It saw the global recovery from the biodiversity crash of the Permo-Triassic Extinction (PTE), and the first appearances of major diapsid reptile clades that would typify Mesozoic ecosystems  –7.However, it has recently been recognized that the morphological diversification of Diapsida in the Triassic was far broader than previously understood –11. A nuDrepanosaurus [As yet, this pattern of convergent morphologies is well established in archosaurs and their close relatives, a possible consequence of overall larger body size and higher preservational potential ,20. Howenosaurus ,27,29. Knosaurus ,31. Hyponosaurus ,30,32, Anosaurus ,33,34 annosaurus ,36. ThesHere, we report on a nearly complete and three-dimensionally preserved skull and partial cervical series of a drepanosauromorph  from the2.Avicranium renestoi, n. gen., n. sp.Diapsida ; Drepano2.1.Avicranium, from aves (Latin for bird) and cranium (Latin for cranium), in reference to the suite of bird-like morphologies present in the holotype skull; renestoi, for Silvio Renesto, who described much of the drepanosauromorph fossil record from Triassic Italy.2.2.AMNH FARB 30834, partial skull and articulated cervical series. Additional drepanosaurid caudal vertebrae and limb fragments are preserved in the block, but are not clearly associated with the individual to which the skull and cervical vertebrae belong.2.3.Effigia okeeffeae by S.J.N. [Coelophysis Quarry . Recovered during preparation of the holotype block of the shuvosaurid pseudosuchian y S.J.N. .2.4.Hypuronector limnaios, Megalancosaurus preonensis and Vallesaurus cenensis  in the complete absence of teeth, a dorsoventrally taller retroarticular process with a triangular shape in lateral view, and cervical neural spines with subequal anteroposterior lengths and transverse widths.Specimens for anatomical comparisons are listed in appendix C. A drepanosaurid diapsid differing from 3.Av. renestoi is very similar to drepanosaurids with cervical series, specifically Drepanosaurus unguicaudatus [Me. preonensis (approx. 27 mm) and substantially larger than the skull of the holotype of V. cenensis (approx. 16 mm).The identification of this specimen as a drepanosaurid is based on its cervical vertebral anatomy. Drepanosaurids possess heterocoelous cervical vertebral centra with saddle-shaped articular surfaces. The prezygapophyseal facets face anteriorly and extend far anteriorly relative to the anterior margin of the centrum. The neural spines are anteroposteriorly short and strongly inclined anterodorsally. In each of these features, caudatus ,31. The 3.1.Av. renestoi exhibits a number of striking similarities to avian theropods . The construction of the orbit differs from that in most Triassic diapsids, in which the cavity is directed anterolaterally [Av. renestoi, the frontal, postfrontal and postorbital all contribute to a transversely broad postorbital septum, which directs the orbital cavity anteriorly (c). The analogous postorbital process in maniraptorans integrates processes of the frontal, squamosal and laterosphenoid [Me. preonensis, based on the tapering rostrum and broadened orbital and temporal regions.The skull of  figures  and 3. Teonensis ,40. Avicd palate e. The coaterally ,41,42. Iteriorly c. The ansphenoid . In mostAv. renestoi from other Triassic reptiles. The contribution to the braincase of the paired frontal and parietal bones is both broad transversely and tall dorsoventrally. This contribution is so prominent that the contributing portion of the frontal is domed dorsally well above the orbital margin (a). This corroborates the hypothesis by [Me. preonensis had an inflated, ‘bulging’ skull roof [Av. renestoi resembles those of Pterosauria and maniraptorans in that the cerebrum is large and broad, occupying much of the anteroventral length of the frontal [Alligator mississippiensis and Gallus domesticus in [Av. renestoi is also transversely broad; the prootics angle strongly medially at their anterior tips to meet dorsolaterally inclined clinoid processes of the parabasisphenoid. A brain enlarged in the way this endocast suggests is otherwise unknown in a Triassic reptile (a). Past studies correlate the enlargement of the brain in pterosaurs and maniraptorans with the adaptation of those taxa to flight—it may be that the enlargement of the drepanosaurid brain followed a similar path to an adaptation to the three-dimensional environments required by arboreality, precision grasping and enhanced stereoscopic vision [Av. renestoi holotype. In our reconstruction, the occipital condyle is slightly posteroventrally inclined relative to the long axis of the skull, in contrast to the strong posteroventral inclination described for Me. preonensis [Av. renestoi may obscure the original shape of the craniocervical articulation.The endocranium preserves some of the most striking departures of the l margin a. This chesis by  that theull roof , p. 251.ull roof –47 and sull roof ,49, taxaticus in ). The an reptile a. Past sc vision ,49,50,53eonensis . However3.2.Captorhinus aguti [Araeoscelis gracilis [Av. renestoi. The quadrate itself is dorsoventrally short and vertically oriented, lacking the posterior embayment in most early saurian reptiles (f) [The anatomy of the suspensorium and other morphologies of the braincase stand in stark contrast to the ‘advanced’ features of the skull roof and rostrum. The squamosal is a dorsoventrally tall, anteroposteriorly broad bone. It exhibits both lateral and posterior laminae that frame the quadrate on those sides, as in archaic eureptiles  and gracilis ). This creptiles f [33,56–iles (f) .Ca. aguti [Ar. gracilis [d). The foramen ovale is extremely large and extends to the ventralmost margin of the braincase. The stapes is massive, with a footplate that entirely fills the foramen and a lateral stem that is larger in all dimensions than the paroccipital process of the opisthotic (d). Foramina ovale and stapedes of this great size are common in early amniotes [Youngina capensis [Mesosuchus browni [Prolacerta broomi [The braincase exhibits a number of traits more commonly found in non-saurian diapsids. The occipital condyle exhibits a deep, posterior depression  across much of its surface and the basal tubera barely extend ventrally below the condyle   and eara broomi ). There a broomi –64.Av. renestoi strongly suggest a plesiomorphic ear. Extant reptiles possess a tympanic membrane framed anteriorly by the concavity of the quadrate, which medially contacts a cartilaginous extracollumella, which in turn meets a very slender, osseous stapes [Av. renestoi suggests the absence of a tympanic membrane. The large foramen ovale with prominent contributions by parabasisphenoid and basioccipital is more common in non-younginiform and non-saurian amniotes, as is the large stapes [Av. renestoi lacks the major osteological correlates of impedance-matched hearing. An atympanic condition occurs in a number of extant lepidosaurs , although these taxa exhibit a slender stapes and a condyle–cotyle articulation between quadrate and squamosal and are widely considered to have undergone secondary loss of external ears. The archaic ear morphology in Av. renestoi, in concert with the other plesiomorphic amniote traits discussed above, contrasts sharply with the comparatively ‘advanced’ condition in most Triassic Sauria [The plesiomorphic diapsid characters of the skull in s stapes ,66. The e stapes ,65,66. Tc Sauria ,60,67.4.Tropidostoma Zone Youngina) dates to the lowermost Upper Permian [In the light of the extensive new data on the cranial anatomy of Drepanosauromorpha provided by AMNH FARB 30834, we integrated the taxon into a phylogenetic analysis focused on terrestrial Permo-Triassic Diapsida and early Sauria .  Permian , suggest Permian –72), as  Permian ).Figure5.Hovasaurus boulei per [These results indicate that drepanosauromorphs represent a deep divergence within Diapsida, earlier than that of crown-group reptiles, but one that persisted through the PTE and radiated deep within the Triassic ,27. A nuulei per ), indicaAv. renestoi holotype, strongly supports the hypothesis that Drepanosauromorpha are non-saurian diapsids. Phylogenetic analyses have long recognized that a number of crown-group reptile lineages (mostly early archosauromorphs) had diverged by the PTE, despite their initial appearance in the fossil record in the Triassic Period [Our revised phylogeny, combined with the extensive character data provided by the c Period ,10,33,63Longisquama insignis and the putative stem-bird Protoavis texensis—which have been used to support the hypothesis that key features of the bird skull evolved very early in the Mesozoic [Av. renestoi instead supports the hypothesis that several bird-like traits first emerged in a Triassic diapsid lineage entirely outside of crown-group reptiles [The general bird-like shape of the drepanosaurid rostrum has long been recognized, owing to complete but crushed specimens from the Upper Triassic of Italy. The three-dimensional preservation of AMNH FARB 30834 adds substantially to the bird-like features of the skull, including the frontated orbits and presumed binocular vision, the absence of teeth, possible fusion of the premaxillae and the inflated endocranium. However, these features occur in conjunction with a strikingly plesiomorphic braincase, suspensorium and postcranial skeleton —featuresMesozoic ,77. ThisMesozoic –80. The reptiles .Av. renestoi differs greatly from that in most Permian and Triassic diapsids. The cerebrum is substantially wider than the olfactory tracts and the endocranium occupies a substantial proportion of the transverse width of the skull, distinctly similar to the brains of maniraptorans , ,82, Av. ans e.g.–85) and  and Av. ans e.g.,86). Man. ManAv. ans (e.g.–89. The romorphs ,39 suggeAv. renestoi, increases the known disparity achieved by terrestrial diapsid reptiles during the Triassic Period and extends the pattern of morphological convergence on later Mesozoic lineages during the Triassic beyond Archosauromorpha into a non-crown-group reptile clade. This and similar discoveries demand constant re-evaluation of the phylogenetic diversity and morphological disparity of fossil groups involved in the recovery from the PTE.This phylogenetic study, in concert with the bird-like characters of the skull of


Influenza A virus (IAV) is a major public health concern, being responsible for the death of approximately half a million people each year. Zoonotic transmissions of the virus from swine and avian origin have occurred in the past, and can potentially lead to the emgergence of new IAV stains in future pandemics. Pulmonary macrophages have been implicated in disease severity in the lower airway, and understanding the host response of macrophages infected with avian influenza viruses should provide new therapeutic strategies.We used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) infected with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ with similar efficiency to the H1N1/WSN virus. While all viruses induced antiviral responses, the H5N3 virus infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses.The LPAI H5N2 and H5N3 viruses are able to infect murine lung macrophages. However, the H5N3 virus was associated with increased expression of pro-inflammatory mediators. Although the H5N3 virus it is capable of inducing high levels of cytokines that are associated with inflammation, this property is distinct from its inability to efficiently replicate in a mammalian host.The online version of this article (doi:10.1186/s12864-017-3803-6) contains supplementary material, which is available to authorized users. Influenza A virus (IAV) represents one of the most important biological threats to human health and the global economy –3. It caIAV normally circulates in wild birds, but it can also infect domestic poultry, pigs, cats, dogs, horses, bats and humans. Although direct zoonotic transmission to humans is not common, interspecies transmission from swine and avian to humans can occurr, and zoonotic transmission can contribute to the emergence of new pandemic viruses –12. BaseAlthough the mechanisms underlying the transformation of LPAI to HPAI is not always clear, evidence suggests that this is a multifactorial process involving the acquisition of specific sequence motifs in several virus proteins e.g. the polybasic cleavage site in the hemagglutinin (HA) protein . HoweverThe murine model of influenza virus infection is commonly used to study the transmission, host adaptation, immune responses and pathogenesis of IAVs (reviewed in ). HoweveIFNγ, IL-4, IL-13, TGFβ), microbes and their products (e.g. LPS), growth factors (e.g. GSF-1 and GM-CSF) and other modulators  . Ta. TaIL-1αIFN). This can be followed by transcription of many antiviral ISGs [GBP1, 2, 3 and 6)) have higher expression levels following infection with the H5N3 virus than the other two isolates  in both viruses and TNF receptor-associated factor 1 (TRAF1) in H5N3 virus infection   or IFNγ) and M2 . M1 macrophages favour glycolytic metabolism, production of reactive oxygen species (ROS) and pro-inflammatory reactions, and are associated with a Th1 response which is essential to combat intracellular infections. The M2 macrophages utilize fatty acid oxidation and mitochondrial respiration and induce anti-inflammatory mediators and are linked to Th-2 response, which is important for extracellular parasitic responses, tissue remodeling, and repair and wound healing. M1 and M2 have a different metabolic profile , 49–53).–53.49–53SLC2A1 and down-regulation of PPARG, ATF3 and TNFAIP812  at 2hpi  and ISGs . Although the levels of expression differed in these studies, several of the inflammatory cytokines and interferon response were detected in both the lung and AECII transcriptome analysis [Influenza virus infection of alveolar epithelial cells type II (AECII) and murine lung tissue showed increased expression of cytokines  and ISGs  were shown in virulent H3N2/HK31 virus infections [CCL2, CXCL2, and TNF-α, CSF3) and interferon responses , which were associated with increased viral load and disease severity [CXCL13, IL-6, IFNG, IFNB1, IRF9, IRF1, and TNF compared with 2009 pandemic H1N1 virus infections. Furthermore, increased expressions of inflammatory cytokines and decreased transcription of genes associated with lipid metabolism correlated with increased disease severity [The H5N3 virus induced higher levels of pro-inflammatory cytokine levels in mouse PMФ compared with the H1N1/WSN and H5N2 viruses. Increased inflammatory cytokine expression in whole mouse lung tissue infected with different mouse-adapted pathogenic/virulent IAV subtypes have been described previously using a transcriptomic approach , 59–65. fections . In addiseverity , 63. TheCCL2 and CCR2+ monocyte-derived cells (monocyte-derived dendritic cells and exudate macrophages) are the predominant cause of immune pathology during PR8/H1N1 virus infection [TRAIL and alveolar epithelial cell (AEC) apoptosis [IL1, IL-6, TNFα, IL-8, MCP-1, IP-10, IFNγ) in patients, in vivo mouse model experiments and in vitro macrophage infections [Lung-resident macrophages have been implicated in disease severity following IAV infection , 66. Thenfection . In addipoptosis . The HPAfections , 72–78.NF-KB or MAPK signaling pathways in a strain dependent manner [Although all the three viruses infected PMФ with similar efficiency, surprisingly the H5N3 virus induced highest inflammatory responses at 24hpi compared to H1N1/WSN and H5N2 viruses. However, we obtained no evidence that the infection of murine macrophages with either of the viruses induced a productive infection . In this context mouse macrophages infected with either of H1N1/WSN, A/HK/483/97 H5N1 (HPAI) or A/X-31/H3N2 viruses showed induced expression of inflammatory cytokines through activation of either t manner –83. Howet manner . Althougt manner , 84. Mort manner , 85–90.TNFα, IL-1αβ, CXCL8, CXCL10, CCL5, IL6, CCL2 and CCL3) production by epithelial cells and macrophages  in RSV-infected PMФ are comparable to the H5N3 virus infection  that are similar to the response that we have observed in RSV-infected PMФ cells . Siiewed in . The levhttp://flusurver.bii.a-star.edu.sg). This information would be of use in surveillance studies which would allow the threat of circulating viruses to be better assessed.Since the murine macrophage cells were equally susceptible to infection with each of the three viruses these differences cannot be explained by reduced infections rates. The H5N3 virus internal genes have been shown to share common ancestry with different subtypes of avian viruses circulating in different geographical locations and environment . This prThe complete mechanism of avian IAV adaptation and pathogenicity to mammalian hosts is not well understood yet. However, it is likely that it involves the HA protein receptor binding specificity and polymerase complex activity . In termARG1 in H1N1 and H5N2 virus-infected PMФ at 2hpi. In M2 activated macrophages increased expression of ARG1 catalyzes polyamine production, which is essential for the synthesis of collagen, tissue remodeling and cell proliferation [NF-kB mediated inflammatory mediators  leads to tissue inflammation and cell death. In addition, the M1 macrophages reprogram glucose metabolism to the glycolytic pathway and the pentose phosphate pathway (PPP), which results in the production of reactive oxygen species (ROS) that mediate tissue disruption [NF-kB mediated inflammatory cytokines and down-regulation expression of pathways related to proliferation and myogenesis. Although the effect of different IAV strains in activation of different macrophages subtypes is not well understood, recently Zhao et al., 2014 suggested that activation of macrophages into M1/M2 phenotype is time and macrophage type dependent [Under normal circumstance, a cell has its own inherent metabolic activity that is responsible for a variety of different physiological processes. Increasing evidence suggests that metabolism under the influence of specific stimuli (e.g. virus infection) is an important determinant for functional phenotype differences in macrophages . There aferation . In linesruption . Similarependent . Continuependent .This analysis demonstrates that LPAI viruses interact with immune-related cells in a virus-specific manner, leading to virus stain-specific changes in pro-inflammatory cytokine expression. The capacity of the avian H5N3 virus to infect mammalian host cells and result in increased expression of inflammatory mediators suggests that this is a unique property that is distinct from other factors, e.g. virus replication in mammals. Since in the natural environment the H5N3 virus can potentially reassort with other circulating influenza viruses, and if such virus is viable this may be able to transfer unique properties that are associated with increased cytokine induction. The identification of sequence motifs in the H5N3 virus that is associated with the increased cytokine induction will be the subject of future studies. This information will allow the identification of sequence motifs that can be used in surveillance studies to assess LPAI viruses which could impose threats to human health in the future.The H5N2 and H5N3 LPAI viruses can infect PMФ similarly to the mouse-adapted H1N1/WSN virus. The global and temporal host gene expression indicated that while there were comparable expression levels of ISG in H1N1/WSN and H5N3 virus-infected cells, increased pro-inflammatory responses were associated with H5N3 virus infection. This systems-based approach provides evidence that increased cytokine induction during influenza virus infection is not a property that is unique to HPAI viruses, but it is also an inherent property of some LPAI viruses. A better understanding of the signals in LPAI viruses that induce host responses to infection should provide insights into the molecular mechanism of pathogenesis. It will also allow a better understanding of the contribution that these viruses play in the emergence of novel influenza strains in epidemics and pandemics.2. The cells were washed with PBS to remove non-adherent cells before used for virus infection. The NP, F4/80-FITC and CD11b antibodies were purchased from Chemicon, Biolegend and MiltenyiBiotec respectively. Cells were infected with each virus using a MOI of 5.H1N1/WSN virus (A/WSN/1933(V-1520)) was purchased from American Type Culture Collection (ATCC). The LPAI isolates H5N2/F118 , H5N3 (A/Duck/Singapore-Q/F119/1997) and H9N2  viruses were obtained from Agri-Food and Veterinary authority of Singapore and have been characterized previously . The 9 tThe cells were on 13 mm cover slides and treated with 4%paraformaldehyde in PBS prior to permeabilization using 0.1% triton X100 in PBS. The cells were then stained with primary anti-NP antibody and FITC conjugated anti-mouse secondary antibody (IgG). The stained cells were mounted on slides using Citifluor and examined using Nikon eclipse 80i fluorescent microscope.Supernatant both from mock-infected or virus-infected PMФ was obtained and centrifuged at 10,000 g for 10 min at 4 °C and the refined supernatant was used for cytokine assay. The cytokines in the supernatant were analyzed according to the Bio-Plex protein array system using the Bio-Plex mouse cytokine 23-plex panel .p-value < 0.05. Differentially expressed genes (DEGs) or probe sets were selected with statistical significant change , ANOVA and the Benjamini-Hochberg False Discovery method  < 0.05) and |Log Fold change (FC)| > 1 (Changes in virus infection in respect to corresponding mock). In the case where a gene is represented by multiple differentially expressed probe sets, the probe with maximum logFC value was selected to construct heat maps. The number of DEGs was determined after summarizing the gene symbols of the differentially expressed using a customized Perl script (by removing redundancies). Venn diagrams showing the overlap of genes in different groups was performed using Venny 2.1 [http://metascape.org/gp/index.html#/main/step1) [PMФ were either mock-infected or virus-infected and at 2 and 24hpi (in H1N1/WSN and H5N2/F118) and 24hpi (H5N3) the cells were harvested at 4 °C using RNA later (Ambion) in PBS buffer, and stored at -80 °C. Three independent experiments were performed for each time point and virus infections. RNeasy mini kit (Qiagen) was used to extract total RNA and double-stranded cDNA was synthesized from 3 μg of total RNA using the GeneChip One-Cycle cDNA synthesis kit . Biotin-labelled cRNA was then synthesized using the GeneChip IVT labelling kit (Affymetrix). After cRNA fragmentation, 15 μg of labelled cRNA was hybridized to the GeneChip Mouse Genome 430 2.0Array (Affymetrix). The arrays were scanned with GeneChip scanner3000 (Affymetrix) after they were washed and stained using the GeneChip Fluidics Station 450 (Affymetrix). Affymetrix.CEL files were generated from GeneChip Operating Software (GCOS) and imported to AltAnalyze 2.1.0 for analysis . Proper enny 2.1 . Function/step1)  and q-van/step1)  and are https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=exkdcksmdhylbcn&acc=GSE86446). Unsupervised hierarchical clustering was used to cluster the DEGs using Gene Cluster version 3 [ Identification of PMФ using ant-F4/80 antibody and ant-CD11b antibody. B) FACS analysis of anti-CD11b and anti-F4/80 stained PMФ. C) Overlap analysis of DEGs in three IAV viruses and RSV ). D) Top 20 significantly enriched pathways of DEGs in IAVs and RSV infected PMФ. E) Unsupervised hierarchical clustering of DEGs at 2 and 24hpi in H1N1/WSN and H5N2 infections. (PDF 230 kb)Additional file 2:A) List of all DEGs with logFC and adjusted p-values B) Expression levels of cytokines in H5N3 compared to H1N1/WSN and H5N2 C) Cytokine expression levels in H5N3 and RSV. (XLSX 233 kb)List of differentially expressed probe sets or genes


Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications. Development of efficient, reliable strategies for long-term preservation of plant virus would largely assist these studies.The present study reported a novel biotechnology allowing cryopreservation of Apple stem grooving virus (ASGV) in living shoot tips. Following cryopreservation by droplet-vitrification or encapsulation-dehydration, about 62–67% of shoot regrowth and 100% of ASGV cryopreservation were obtained. Although shoot proliferation and virus concentration were reduced in cryopreserved diseased shoots after 8 weeks of shoot regeneration, continuous subculture for 4 times (16 weeks) increased shoot proliferation and virus concentration to comparative levels as those produced by shoot tip culture (as a control to shoot tip cryopreservation). Cryopreserved ASGV was efficiently transmitted to a woody plant by micrografting and to a herbaceous indicator by mechanical inoculation. Gene sequencing in three fragments of ASGV genome including coat protein and movement protein showed that cryopreserved ASGV shared 99.87% nucleotide identities with shoot tip culture-preserved virus, indicating cryopreserved virus is genetically stable.The present study demonstrates ASGV, a representative virus that can infect meristematic cells of shoot tips, can be efficiently cryopreserved in shoot tips. To the best of our knowledge, this is the first report on plant virus cryopreservation in living tissues, and has great potential applications to long-term preservation of plant viruses.The online version of this article (10.1186/s13007-018-0312-9) contains supplementary material, which is available to authorized users. Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications such as antigen preparation for virus detection by immunology-based methods , productCryopreservation, i.e. storage of living cells, tissues and organs in extra low temperature, usually that of the liquid nitrogen (LN), was first proposed with a motive for long-term preservation of plant genetic resources by Sakai , is at pVirus is unevenly distributed in plants: virus titer decreases as the distance increases from apical dome (AD), thus resulting in low virus titer or even virus free area in the very top parts of AD . In shooActinidia isolate), with obvious cross banding and helical symmetry [Malus crops. It also attacks other fruit crops including Pyrus, Citrus and Actinidia, as well as a number of important ornamental crops [Apple stem grooving virus (ASGV) in shoot tips of ‘Gala’ apple. Cryopreserved virus was also tested for its infectious ability to infect apple (a woody plant) by micrografting and Nicotiana benthamiana (a herbaceous indicator) by mechanical inoculation. Gene fragments of cryopreserved ASGV genome were sequenced.ASGV, a type member of the genus Capillovirus, is a single-stranded RNA virus, and the virus particles are flexuous filaments 620 nm long (apple strain), 650 nm long (citrus strain) and 680 nm long  were transferred after 12 and 36 h after the initial post-thaw culture to minimize oxidation reactions. Shoot tips cryopreserved by encapsulation-dehydration were extracted from the beads after 1 week of post-culture and then recovered directly on medium. Shoot regrowth was expressed as the percentage of surviving shoot-tips that formed normal shoots (≥5 mm in length) with at least three fully-opened leaves 8 weeks post-thaw culture. Subculture was performed once every 4 weeks. Regrown shoots (1.0 cm long) were transferred onto fresh SMM for shoot proliferation and subcultured once every 4 weeks. Shoot length and number of shoots proliferated per explant were measured in 1, 2 and 4 times of subculture.Culture of shoot tips (the same size as used for shoot tip cryopreservation) excised from ASGV-infected shoots was used as control of virus cryopreservation. Shoot tips were cultured on SMM for shoot regrowth in the light condition, as described for in vitro stock shoots. Shoots regenerated after 8 weeks of culture were used for shoot proliferation, the same as for shoots regenerated from cryopreservation.Shoots regenerated from cryopreserved shoot tips that had been subcultured for 4 times 16 weeks) were used for virus transmission by micrografting upon the healthy in vitro shoots of ‘Gala’ apple. Micrografting was conducted, as described by Hao et al. . Shoot s6 weeks wMicrografts were taken at 0, 3, 7 and 14 days of micrografting and used for histological observations, as described by Hao et al. . In brieN. benthamiana were sown in 8-cm pots containing peat  and grown for production of seedlings in a net-proof greenhouse under a 16-h photoperiod with a light intensity of 225 µmol/s m2 provided by Philips LED lamps at 24/20 °C of day/night temperatures. Virus transmission by mechanical inoculation was performed, according to Clover et al. [N. benthamiana seedlings were mechanically inoculated with ASGV. ASGV-induced symptoms were observed at day 21 of inoculation. Samples inoculated with only sodium phosphate buffer without virus were used as control.Seeds of r et al. . In brieVirus status was assessed by RT-PCR three times in the present study, according to Li et al. . The firRelative mRNA expression levels of ASGV were analyzed by qRT-PCR in the diseased shoots regenerated from cryopreserved shoot tips after 1 (4 weeks), 2 (8 weeks), 4 (16 weeks) times of subculture of shoot proliferation. qRT-PCR was performed, according to Sun et al. . Total REscherichia coli strain JM109 was transformed. For each fragment, five clones were sequenced in both orientations to confirm the consensus sequence using an Applied Biosystems 3730 DNA Analyzer . Where polymorphic was encounter, at least five more clones were sequenced to obtain the consensus sequence. Sequence alignment analysis was performed using the program Vector NTI 11.5.1 .ASGV cryopreserved by droplet-vitrification and preserved by shoot tip culture was used for gene fragment sequencing. Three gene fragments of ASGV genome with their nucleotides positioning from 235 to 756, 1210–2445 and 4310–6364 were cloned and sequenced, according to Zhao et al. . Choice In experiments of cryopreservation, shoot proliferation and micrografting, 10 samples were included in each treatment of three replicates. The whole experiment was repeated three times. Thus, each experiment had 30 samples in three replicates. Data are presented as mean ± SE. Statistical differences among means were assessed by analysis of variance (ANOVA) and significant difference (P < 0.05) was analyzed by Tukey’s multiple range test. For virus detection by RT-PCR and RT-qPCR, 20 samples randomly selected from the three independent experiments were included in each treatment. Ten samples from each treatment were used for gene fragment sequencing.Surviving shoot tips started to show green color at about 7 day of post-thaw culture following cryopreservation Fig. a. SurvivWith RT-PCR, amplified segments of 524 bp were detected in in vitro diseased stock shoots that were used in the experiments for ASGV cryopreservation, while no such bands were detected in the healthy samples  [Pelargonium flower break virus (PFBV) [Pelargonium line pattern virus (PLPV) [Frequencies of shoot regeneration of cryopreserved shoot tips and virus cryopreservation, as well as virus concentration preserved in the samples, and proliferation of virus-infected shoots regenerated from cryopreserved shoot tips, are the important factors determining virus cryopreservation efficiency. In the present study, more than 62–67% of cryopreserved shoot tips regrew into shoots. Theoretically, the samples can be stored in LN for an indefinite duration and maintain their recover ability, once they survive freezing in LN , 44. PreMalus . In the s (RBDV) , Pelargos (PFBV) , Pelargos (PLPV)  and ASGVs (PLPV) –23. Obvis (PLPV) , who atts (PLPV) . ResultsN. benthamiana (a herbaceous indicator) by mechanical inoculation. Histological observations on micrografting showed micrografts developed normally, as noted in micrografting of virus-infected grapevine leafroll-associated virus-3 [Grapevine leafroll-associated virus-3. Virus mechanical transmission showed typical symptoms of ASGV on the leaves of N. benthamiana after 21 days of inoculation. These results were consistent with those reported by De [ASGV can be transmitted by grafting and mechanical inoculation . In the  virus-3 . Virus t virus-3 , who useed by De , Hirata ed by De , Lovisoled by De  and Bhared by De , 52 for Like plant germplasm, genetic stability is a major concern in virus preserved by any methods. When biological samples are frozen in LN, all cell division and metabolism are arrested, and the samples can be stored in LN, theoretically for an unlimited time period, thus limiting the opportunities for changes in the genetic integrity of stored materials , 54. HowSweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV) [Cucumber mosaic virus (CMV) [Cryopreservation can eradicate viruses that are not able to infect meristematic cells of shoot tips and frequencies of virus eradication varied largely from 100% in  (SPFMV) , to 30% us (CMV) . This meus (CMV) –30. Cellus (CMV) , 57, 58.In conclusion, the present study demonstrates ASGV, a representative virus that can infect meristematic cells of shoot tips, can be efficiently cryopreserved in shoot tips and cryopreserved ASGV can effectively be transmitted to woody plant by micrografting and to herbaceous indicator by mechanical inoculation. Cryopreserved ASGV is genetically stable. It is well-known that a number of viruses and almost all viroids can infect meristematic cells of shoot tips –27. ThusAdditional file 1: Table S1. Primers used for ASGV detection by RT-PCR, RT-qPCR and gene amplification in ASGV cryopreservation of virus-infected shoots of ‘Gala’ apple the present study. Figure S1. ASGV detection by RT-PCR for ASGV in in vitro stock shoots, and shoots regenerated from two cryopreservation methods and shoot tip culture in ‘Gala’ apple (A) and RT-PCR analysis for EF-1α gene as a reference (B). Figure S2. Gene fragments of ASGV genome used for gene sequencing in cryopreserved virus in in vitro shoots of ‘Gala’ apple. Figure S3. Comparison of gene fragments of A, B and C of ASGV genome preserved between cryopreservation and shoot tip culture.


Plant–virus interactions based-studies have contributed to increase our understanding on plant resistance mechanisms, providing new tools for crop improvement. In the last two decades, RNA interference, a post-transcriptional gene silencing approach, has been used to induce antiviral defenses in plants with the help of genetic engineering technologies. More recently, the new genome editing systems (GES) are revolutionizing the scope of tools available to confer virus resistance in plants. The most explored GES are zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats/Cas9 endonuclease. GES are engineered to target and introduce mutations, which can be deleterious, via double-strand breaks at specific DNA sequences by the error-prone non-homologous recombination end-joining pathway. Although GES have been engineered to target DNA, recent discoveries of GES targeting ssRNA molecules, including virus genomes, pave the way for further studies programming plant defense against RNA viruses. Most of plant virus species have an RNA genome and at least 784 species have positive ssRNA. Here, we provide a summary of the latest progress in plant antiviral defenses mediated by GES. In addition, we also discuss briefly the GES perspectives in light of the rebooted debate on genetic modified organisms (GMOs) and the current regulatory frame for agricultural products involving the use of such engineering technologies. Viruses are well-known to be one of the major concerns for agricultural production and food security throughout the world. It is estimated that viral agents are responsible for the half of emerging diseases reported in plants . The conTobacco mosaic virus in Nicotiana glutinosa led to the initial understandings on plant viral immune responses and the introgression of resistance genes from wild to cultivated plants . The RNAi is a biological mechanism whereby small RNA molecules, such as small interfering RNA (siRNA) or microRNA (miRNA), can regulate gene functions via post-transcriptional gene silencing. A critical breakthrough was the demonstration that double-strand RNA (dsRNA) molecules trigger the RNAi pathway to regulate gene expression in  elegans . This mo elegans . The RIS elegans . In plan elegans . Althoug elegans . The app elegans , and cur elegans . However elegans . Besides elegans . Therefo elegans . These aAgrobacterium infection and ballistic bombardment (Agrobacterium-mediated transformation is based on the transfer and insertion of a given DNA sequence into a plant genome by plasmids Ti (tumor-induced) or Ri (rhizogenic) from the bacterium  in a targeted gene , meganucleases, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs) and more recently the clustered regularly interspaced short palindromic repeats (CRISPR) along with the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9).Genome editing systems approaches against plant viruses have been mainly developed using ted gene . After tted gene . Thus, Dted gene . Four maMeganucleases or homing endonucleases are encoded by introns and inteins that recognize DNA sequences between 12 and 42-base-pair (bp) in length, unlike restriction enzymes . The megFokI restriction enzyme  of a zinc-finger protein with the DNA cleavage domain of the n enzyme . FokI woion site . A ZFN iion site . An effeion site .FokI  and the non-specific cleavage domain of the enzyme FokI . TALEs arocesses . Once TArocesses . TALE prrocesses . The DNArocesses . Thus, tStreptococcus thermophilus the bacteria are able to generate virus-resistant mutants through integration of viral genome sequences into the CRISPR loci in association with cas genes expression. Among CRISPR/Cas systems, the CRISPR/Cas9 from S. pyogenes is the most studied model (cas9 protein containing two nuclease domains (RuvC and HNH) that cleave both strands of the DNA target leading to DSBs and site mutations, and (ii) a guide RNA (gRNA) whose role is direct cas9 protein to the DNA target. A gRNA is composed by two different RNA molecules: a CRISPR RNA (crRNA), which contains complementary sequences to the DNA target, and a trans-activating crRNA (tracrRNA) involved in processing of precursor crRNA molecules to a mature crRNA , which was also able to direct Cas9 to the DNA target . After sAs mentioned above, GES such as ZFN, TALEN, or CRISPR-Cas9 are provided of DNA-binding and catalytic domains. Meanwhile, TALE and artificial zinc finger protein (AZP) have DBDs and lack of catalytic domains. TALE and AZP can be engineered to prevent viral multiplication by blocking specific DNA sites in the viral genomes, which are essential for DNA-binding proteins of the virus and subsequent interactions with the replication machinery of host cell.Table 1 are summarized the several genome targeting technologies that have been explored to provide potential control against plant viruses. It is important to note that the most evaluated species belong to the families Geminiviridae and Potyviridae. According to the latest report of the International Committee on Taxonomy of Viruses (ICTV), Geminiviridae and Potyviridae families represent the two largest groups of plant viruses containing 326 and 195 species, respectively (In ectively . Hence tin planta targeting a 19-bp fragment in the intergenic region (IR) of Beet severe curly top virus . BSCTV, like all members of family Geminiviridae, has an IR containing a binding site recognized by the replication initiator protein (Rep) to initiate viral replication  by blocking the Rep binding site of TYLCV  in transgenic A. thaliana plants carrying an AZP which was able to recognize and block promoter sequences of RTBV  was developed to target the AC1 gene of the virus that codes the Rep protein , another begomovirus, showing an inhibition of the replication of the virus and, thus, suggesting a possible resistance strategy to be broadly used against begomoviruses.The first efforts to introduce a viral inhibition factor in plants, through zinc finger technology, were carried out in the mid-2000s. lication . BSCTV rSCTV Rep . In vitrof TYLCV ; however of RTBV . AZP for of RTBV . Unlike  protein . In thatTomato leaf curl Yunnan virus (TLCYnV) and Tomato yellow leaf curl China virus (TYLCCNV). Transgenic plants of N. benthamiana carrying the TALEs displayed resistance to TbCSV and TYLCCNV, while the resistance to TLCYnV was partial. Although a broad-spectrum approach for resistance to a virus group is desirable, it seems difficult to predict a unique TALE system to control a large group of virus like geminiviruses. TALE technologies including nuclease domains (TALEN) have not been still reported against plant viruses. For human viruses, the potential antiviral applications of TALEN have been explored for several viruses such as Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), however, the generic challenges for gene therapy  remain to be solved , suggesting a possible interference of the secondary structure on the sgRNA-Cas9 cleavage. Despite that, viral load was reduced probably by blocking of the Rep binding site 4E gene of A. thaliana, which were efficient to confer resistance to the potyvirus Turnip mosaic virus (TuMV) In the light of these studies, CRISPR-Cas9 systems could be developed to target host genes coding for other members in the family of plant translation factors such as eIF4G, eIF(iso)GE, eIF4A-like helicases, eIF3, eEF1A, and eEF1B that are also identified to interplay with protein and viral RNAs . Although the members of the plant virus families Geminiviridae and Nanoviridae are composed of single-stranded DNA (ssDNA), they also contain replicative intermediate forms of dsDNA which can be targeted by GES. The other plant virus family possessing DNA genomes is Caulimoviridae. Unlike geminiviruses and nanoviruses, the caulimoviruses have a dsDNA genome. Currently, there are 432 virus species that belong to the DNA virus families Geminiviridae, Nanoviridae, and Caulimoviridae (Metaviridae and Pseudoviridae that infect plants could be directly targeted by GES. Metaviruses and pseudoviruses are reverse transcribing RNA viruses. Although their genomes are single-stranded RNA (ssRNA), they possess replicative dsDNA forms (Genome editing systems are known to bind to double-stranded DNA (dsDNA) and subsequently introduce a DSB in a sequence-specific manner. Hence, the first studies using genome targeting or genome editing platforms for plant viruses aimed to control DNA viruses  RNase domains  gene, which is related with mildew powder susceptibility (Agaricus bisporus)  . In geneA. thaliana lacking the CRISPR/Cas9 system after two generations. Insertion of point mutations are also feasible using mutagenic agents. For example, using the technique TILLING  point mutations in tomato eIF4 were induced by ethyl methanesulfonate to confer immunity to two potyviruses  are the most economically important viruses for banana production worldwide . The GMO definition according to the Cartagena Protocol on Biosafety and the European Union was mainly conceived to distinguish products obtained by conventional plant breeding technologies and those obtained by recombinant DNA technologies . Developyviruses . Besidesyviruses . In anoteign DNA . Such aporldwide . InteresCurrently there is a growing worldwide debate about to how to regulate research and use of plant produced with the novel genome editing technologies . In the Geminiviridae and Potyviridae. Among these technologies, CRISPR-Cas9 has been already explored for most of the viruses tested, despite the fact that it is the more recently developed GES. This fact might be explained by the simplicity of CRISPR-Cas9 designing, which can be adopted by standard biotechnological laboratories. However, off-targets effects (possible breaks in non-targeted DNA sites) continue as a major concern for application of CRISPR-Cas9 technology. A next generation sequencing (NGS) approach can be useful to provide a compelling profile of off-target cleavage sites for a given GES , Tale and CRISPR-Cas9 technologies against plant viruses have been done for at least 14 different species, mainly in the families iven GES . Likewisiven GES . Beyond GR wrote the draft version of manuscript and CB reviewed the manuscript providing substantial contributions. Both authors read and approved the final version of the work.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.


Culex quinquefasciatus and Culex tritaeniorhynchus in Kwale, Kenya and provinces of Hubei and Yunnan in China. Our results showed that viromes from the three locations were strikingly diverse and comprised 30 virus families specific to vertebrates, invertebrates, plants, and protozoa as well as unclassified group of viruses. Though sampled at different times, both Kwale and Hubei mosquito viromes were dominated by vertebrate viruses, in contrast to the Yunnan mosquito virome, which was dominated by insect-specific viruses. However, each virome was unique in terms of virus proportions partly influenced by type of ingested meals . The dominant vertebrate virus family in the Kwale virome was Papillomaviridae (57%) while in Hubei it was Herpesviridae (30%) and the Yunnan virome was dominated by an unclassified viruses group (27%). Given that insect-specific viruses occur naturally in their hosts, they should be the basis for defining the viromes. Hence, the dominant insect-specific viruses in Kwale, Hubei, and Yunnan were Baculoviridae, Nimaviridae and Iflaviridae, respectively. Our study is preliminary but contributes to growing and much needed knowledge, as mosquito viromes could be manipulated to prevent and control pathogenic arboviruses.Many blood-feeding arthropods are known vectors of viruses that are a source of unprecedented global health concern. Mosquitoes are an integral part of these arthropod vectors. Advancements in next-generation sequencing and bioinformatics has expanded our knowledge on the richness of viruses harbored by arthropods. In the present study, we applied a metagenomic approach to determine the intercontinental virome diversity of  Plasmodium spp., filarial nematodes, Zika virus, dengue virus, Rift Valley fever virus and yellow fever virus. The diversity and richness of mosquito flora is only beginning to be understood following the application of next-generation sequencing (NGS) and vector-enabled metagenomics (VEM). These advanced molecular platforms have revealed that in addition to known pathogens, mosquitoes harbor a large proportion of organisms whose pathogenicity in vertebrate hosts is not yet known. Previous metagenomic studies have revealed that mosquitoes harbor a highly diverse microbiome composed of bacteria, protozoa, and viruses, of which some are specific to arthropods, protozoans, and plants [Global health security is increasingly threatened by emerging and re-emerging pathogens whose consequences to human and animal health are dire and devastating to economies. Most of these pathogens have their origin in diverse hosts, mainly wildlife, and are vectored by multiple blood-feeding arthropods. Part of the global health challenge is that some of these arthropods are widely distributed and highly efficient vectors for many pathogens. Specifically, the mosquito is one blood-feeding arthropod, known to feed on myriad hosts, and is an efficient vector for a multitude of virulent pathogens such as d plants ,2,3,4. TThere is a developing specific interest to understand mosquito viromes, particularly in terms of virus diversity and composition. This arises from the fact that most mosquito-borne viruses bear significant threat to public and animal health, whereas a large proportion (<70% amino acid identity) of recovered viruses are of unknown pathogenicity ,6. SurveTogaviridae, Flaviviridae, Rhabdoviridae, Bunyaviridae and Mesoniviridae), they constitute a significant component of mosquito viromes. In addition, it seems that each virome is dominated by particular viruses, which hypothetically may be transient, but significant in distinguishing or describing diverse viromes. For example, the virome of Culex spp. in Zambezi, Mozambique was dominated (88.5%) by insect-specific viruses, of the Iflaviridae family [Culex tritaeniorhynchus virome in Hubei province also showed a similarly high dominance (88%) of insect-specific viruses [Besides viruses that can replicate in vertebrates, mosquito viromes seem to harbor a large proportion of viruses that naturally infect insects and can replicate only in insect cell lines in vitro. Even though these insect-specific viruses seem not to replicate in vertebrates , some ofe family . In Chin viruses .Aedes albopictus first made its incursion to South Africa through trade in old car tires [Culex mosquitoes in specific sites in China and Kenya. The Culex mosquito is an efficient vector for several arboviruses such as West Nile virus, Rift Valley fever virus, and Japanese encephalitis, as well as parasitic bancroftian filariasis. African mosquito virome studies are in their nascent stage, with few exploratory investigations, though outcomes from such studies show remarkable diversity and composition [Culex spp. in both Kenya and China.Increased frequency and speed of human travel and commercial trade has been implicated in the expansion of mosquito vectors and their pathogens from indigenous habitats to new areas. The well-known ar tires  and has position ,13. Our The study areas in Kenya were located in the Kwale and Kilifi counties, along the Indian Ocean coastline. The study areas in China were located in the Hubei province in south-central china and the Yunnan provinces in south-western China.http://www.opendata.go.ke). The region has experienced several arbovirus outbreaks due to its high human population together with high inflow and outflow of visiting tourists. Also, the close vector/human/wildlife proximity might be a contributing factor to the emerging outbreaks in the region.Kwale county is located on the south-eastern part of Kenya along the shores of the Indian ocean . It lieshttp://www.kilifi.go.ke). There is a precipitation difference of 246 mm between the driest and wettest months. The annual temperature variation is 3.8 °C. Due to its proximity to the Indian Ocean, tourism and fishing are the main economic activities hence experiencing a high human traffic.Kilifi county lies along the coastal belt, in the south-eastern part of Kenya. The region receives an average annual rainfall of 900–1100 mm. The annual temperatures ranges from 21 °C to 30 °C in the coastal belt but rise to maximum of 34 °C in the hinterland (Source: http://en.hubei.gov.cn).Hubei Province is located in south-central China . It has Yunnan Province is located in the south-western part of China  and it h® Woodstream Corp., Brampton, ON, Canada) from outdoor sites between dusk and dawn, in the months of January to February 2017. In China, adult mosquitoes were sampled from two provinces, namely the Hubei province  and Yunnan province   . In Hube® Stranded Total RNA Sample Preparation kit  following the manufacturer’s instructions. Briefly, ribosomal RNA was removed from total RNA using Ribo-Zero rRNA removal beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under 94 °C for 8 min. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process, the addition of a single “A” base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer  and validated by Agilent 2100 bioanalyzer  to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq 2500 . The library construction and sequencing was performed at Shanghai Biotechnology Corporation.The mosquito pools were homogenized in 1 ml Roswell Park Memorial Institute medium (RPMI) and the homogenate filtered through a 0.22 μM polycarbonate filter membrane  to exclude bacteria and other debris. RNA was extracted from 140 μL of homogenate using the QIAamp viral RNA extraction kit according to manufacturer’s instructions . The RNA integrity was checked using the Agilent bioanalyzer 2100  and further purified using the RNA clean XP kit . Strand-specific libraries were prepared using the TruSeqCulex genomes with Bowtie2 [Bioinformatics analysis was performed as described by Guillaume et al.  but with Bowtie2 . The seq Bowtie2 . These d Bowtie2 . An E-va Bowtie2 .Taxonomic classification of identified viruses was done by conducting BLASTn and BLASTx on National Center For Biotechnology Information (NCBI) virus database and the obtained Blast XML files were imported into MEGAN6 program  and defa2O to supplement the system. Thermal cycling was initiated with a denaturation step at 94 °C for 5 min; followed by 35 cycles of 94 °C for 30 s, 58–60 °C 30 s and 72 °C for 1 min; and a final extension at 72 °C for 10 min. The PCR products were extracted with a Gel extraction kit  and sequenced at Tsingke Biological Technology. The primers used are presented in Analyzed viral sequences were used to design specific PCR primers to confirm the presence of some selected viruses from the original RNA. PCR amplifications were carried out in a total volume of 25 μL consisting of 2 μL of template (cDNA), 0.25 μM of forward and reverse primers, 12.5 μL of PrimerSTAR Max Premix 2× , and Milli-Q ddHCulex spp. mosquitoes in sites within Kilifi County except sites in Kwale County where we dominantly trapped Culex quinquefasciatus. In China, we dominantly trapped Culex tritaeniorhynchus in all the sites within Hubei and Yunnan provinces. We sequenced 47 mosquito pools from Kwale, 100 pools from Hubei, and 100 pools from Yunnan. Mosquito samples from Kwale, Kenya generated a total of 21,747,508 raw reads. Samples from Hubei and Yunnan provinces, China had 6,714,707 and 36,277,174 raw reads, respectively. The average sequence reads lengths were 150 bp, 125 bp, and 398 bp for Kwale, Hubei and Yunnan respectively  being most dominant, followed by Herspesviridae (8.17%). Among the invertebrate specific viruses, Baculoviridae was the notable virus family, whereas Partitiviridae (0.6%) was the leading plant virus. Kwale had a notable proportion of protozoan viruses, which were dominated by Pandoravirus salinus and Pandoravirus dulcis (unclassified viruses). These viruses were not identified in samples from China.In Kwale, vertebrate, invertebrate, plant and protozoan viruses were the most abundant . The virHerpesviridae (30.16%) was the dominant vertebrate virus in the virome of Hubei followed by Adenoviridae (14%). Nimaviridae (3.6%) was the leading virus family in the invertebrate host group, while Genomoviridae (9.4%) was leading in part of plant viruses from Hubei.In the Hubei province, vertebrate, invertebrate, plant and unclassified viruses were the most abundant . HerpesvIflaviridae, Dicistroviridae, and Mesoniviridae, and some belonged to the unclassified virus groups. Our results showed that the Wuhan mosquito virus (unclassified viruses) and Culex tritaeniorhynchus rhabdovirus (Rhabdoviridae) were the most common virus species. In addition, Yunnan had a higher proportion of unclassified viruses (27.17%) compared to Kwale and Hubei. Under vertebrate viruses, the family Reoviridae (24.6%) was the most conspicuous group and it was dominated by the Banna virus (33%) and Kadipiro virus (11.6%) (The virome of mosquito in the Yunnan province was dominantly composed of invertebrate-host related viruses (58.7%)  of which (11.6%) .Flaviviridae  and Herpesviridae  as well as the group of unclassified viruses .In the three study sites, the three most abundant virus families were Flaviviridae, both Hubei (2.7% reads) and Kwale (1.6% reads) had flaviviruses that infected vertebrates and also some insect flaviviruses. Specifically, in Kwale we identified the dengue 2 virus (77 reads) which was further confirmed by PCR. Culex flavivirus (CxFV) was the only insect-specific flavivirus identified in Kwale. Yunnan Province exclusively had insect-specific flaviviruses (2.5%), mainly dominated by the Quang Binh virus (QBV). In Hubei province, flavivirus was similarly dominated by QBV.Under the family Mimiviridae (protozoan-host) was present in all the three study sites, while Totiviridae (protozoan-host) was present in Yunnan and Kwale mosquitoes. Mycoviruses, bacteriophages, and environmental viruses contributed a significantly small percentage in all the three study sites.Additionally, the family Emerging studies on mosquito viromes depict a highly diverse community of viruses whose existence, role, and transmission are less understood. Our results are thus consistent with previous studies as they revealed a rich diversity of both known pathogenic groups of viruses to humans and animals as well as viruses specific to other organisms . Given that mosquito trappings in the three locations in Kenya and China were carried out at different times, our findings should be interpreted with that in mind. From our findings, Kwale, Kenya and Hubei, China  were comAdenoviridae, Anelloviridae, Circoviridae, Coronaviridae, Flaviviridae, Herpesviridae, Papillomaviridae, Reoviridae, Retroviridae, Togaviridae and Rhabdoviridae. These virus families are of medical and veterinary importance and some have been identified as part of mosquito viromes elsewhere [Across the three locations, the mosquito viromes harbored a total of 30 virus families and one general group of unclassified viruses . The virlsewhere . Across Reoviridae) had the most abundant viral reads in this set , Banna virus and Kadipiro virus  in the Kwale mosquito virome. The virus causes variable symptoms ranging from mild febrile illness to the more severe dengue hemorrhagic fever, which can progress to a fatal form, dengue shock syndrome (DSS). Aedes aegyptii and Aedes albopictus are the two competent vectors of the virus worldwide [Culex mosquitoes ingested the virus from viremic hosts. The Kenyan coastal towns, which include Kwale, have experienced intermittent outbreaks due to dengue 2 in the past and presence of the virus suggests continuous low-level circulation.Although not among the top ten virus species , but ratorldwide ,60,61; hCulex tritaeniorhynchus is the main vector of the endemic Japanese encephalitis virus (JEV) and other zoonotic viruses like Banna virus and Kadipiro virus in Yunnan, Hubei and other provinces of China [Culex univittatus from the Rift Valley [Iflaviridae, Dicistroviridae and Mesoniviridae as the most common, which is consistent with virome diversity and proportions of Culex spp. in Zambezi, Mozambique and Hubei, China [Culex mosquito viromes in Zambezi and Hubei were dominated (up to 88%) by the insect-specific viruses, Iflaviridae [Nimaviridae, that is usually composed of a single genus, Whispovirus, known to be of global occurrence and fatally infects crustaceans [Baculoviridae, a virus family that is known to infect diverse invertebrates but has also been reported to cause epizootics in field populations of Culex spp. [Culex flavivirus (CxFV), a virus that has previously been isolated across the world.It is worth noting that of China ,62,63,64of China ,66,67. It Valley , wild bit Valley  and in tt Valley , We did i, China . Interesaviridae . In addistaceans . In contlex spp. . Specieslex spp. . In addiTymoviridae—a group that constitutes globally distributed viruses—that causes significant losses in crops due to plant diseases [Culex originated Tymoviridae-like virus (CuTLV) (Tymoviridae), isolated from Culex spp. in China, showed cytopathic effect on mosquito cell lines, implying a potential for being vectored by mosquitoes [In the present study, we identified a notable proportion of plant-specific viruses across viromes in the three locations . We idendiseases . Mosquitsquitoes .Baculoviridae, Nimaviridae and Iflaviridae, respectively, and hence such dominant viruses might have a role in the component community of the host. Our study is preliminary but due to emerging theories that suggest that manipulation of specific components of mosquito viromes could yield potential agents for control and prevention of pathogenic arboviruses and also the global emerging trends of mosquito-borne diseases, more studies are still necessary to uncover the global patterns of composition and diversity of mosquito viromes.In summary, mosquito virome studies are very few, implying that there is an enormous diversity of viruses yet to be uncovered. Our study was preliminary but it revealed that at a larger habitat scale, geolocation as well as the sampling time period are important factors that could enhance recovery of a greater diversity of mosquito viruses. We determined that each virome was unique in terms of virus proportions partly influenced by type of ingested meals . Although vertebrate and other viruses ingested from other hosts are significant components of the virome, we suggest that since insect-specific viruses occur naturally in their hosts, they should be the basis for defining the mosquito virome. For instance, in this study, the dominant insect-specific viruses in Kwale, Hubei, and Yunnan were


Flaviviridae, Flavivirus) is comprised of Spondweni virus (SPONV) and Zika virus (ZIKV), which are mosquito-borne viruses capable of eliciting human disease. Numerous cases of ZIKV sexual transmission in humans have been documented following the emergence of the Asian genotype in the Americas. The African ZIKV genotype virus was previously implicated in the first reported case of ZIKV sexual transmission. Reports of SPONV infection in humans have been associated with non-specific febrile illness, but no association with sexual transmission has been reported. In order to assess the relative efficiency of sexual transmission of different ZIKV strains and the potential capacity of SPONV to be sexually transmitted, viral loads in the male reproductive tract and in seminal fluids were assessed in interferon α/β and –γ receptor deficient (AG129) mice. Male mice were inoculated subcutaneously with Asian genotype ZIKV strains PRVABC59 , FSS13025 , or P6-740 ; African genotype ZIKV strain DakAr41524 ; or SPONV strain SAAr94 . Infectious virus was detected in 60–72% of ejaculates collected from AG129 mice inoculated with ZIKV strains. In contrast, only 4% of ejaculates from SPONV-inoculated AG129 males were found to contain infectious virus, despite viral titers in the testes that were comparable to those of ZIKV-inoculated mice. Based on these results, future studies should be undertaken to assess the role of viral genetic determinants and host tropism that dictate the differential sexual transmission potential of ZIKV and SPONV.The Spondweni serogroup of viruses ( in utero transmission have also been described. Due to the close genetic identity of Zika and Spondweni viruses, the herein reported study used a mouse model to assess the sexual transmission capacity of Spondweni virus in comparison to recent outbreak Zika strains and older Zika virus strains. In this model, all Zika strains were shed in seminal fluids from infected males. However, the percentage of ejaculates that contained infectious virus was significantly lower for Spondweni-infected males than Zika-infected males. Thus, sexual transmission potential is conserved among Zika viruses and not likely to fully explain the magnitude and dynamics of the recent outbreak in the Americas. In addition, sexual transmission potential should be further evaluated for Spondweni virus. Virus-specific differences in rates of shedding in seminal fluids will inform future studies on the viral determinants of sexual transmission.The Spondweni serogroup of viruses, which includes Zika virus and Spondweni virus, are mosquito-borne viruses that can cause disease in humans. During the recent outbreak of Zika virus in the Americas, sexual transmission and  Flaviviridae, Flavivirus). Since the initial isolation of SPONV in 1952 CATAGGACTGCTGGTGGTGA[TAM]; Forward primer (5’ AACCAAGACCGACATTGACA 3’); Reverse primer (5’ CACTCTTGCCAGAAACCACA 3’). All real-time assays were performed by using the QuantiTect Probe RT-PCR Kit  with amplification in the Bio-Rad CFX96 Touch real-time PCR  following the manufacturer’s protocol. The detection limit for this assay was 3 log10 RNA copies/mL (or 1.5 log10 RNA copies/ejaculate).RNA was extracted from seminal fluid using the MagMax Viral RNA Isolation kit (Ambion), as described previously , with thSurvival curves were compared using a log-rank (Mantel-Cox) test. Viral titers in tissues and ejaculates and testicular weights were compared using ANOVA, and viral titers in serum were compared using multiple t-tests with a Holm-Sidak correction for multiple comparisons. Proportions of ejaculates containing infectious virus were compared using Fisher’s Exact Test. Statistical tests were performed in GraphPad Prism.All experiments involving mice were approved by institutional animal care and use committee (IACUC) at the Division of Vector-Borne Diseases, Centers for Disease Control and Prevention under protocol 16–013. All protocols and practices for the handling and manipulation of mice were in accordance with the guidelines of the American Veterinary Medical Association (AVMA) for humane treatment of laboratory animals.


In September 2014, a single fatal case of Marburg virus was identified in a healthcare worker in Kampala, Uganda. The source of infection was not identified, and no secondary cases were identified. We describe the rapid identification, laboratory diagnosis, and case investigation of the third Marburg virus outbreak in Uganda. Filoviridae, which also includes Ebola viruses). The disease was first discovered in 1967 in Marburg and Frankfurt, Germany, after laboratory workers were infected from monkeys imported from Uganda (Marburg virus disease (MVD) is caused by Marburg virus , the host reservoir of MARV . The patient, a 30-year-old man, was a radiographer who worked at Mengo Hospital and part-time at Mpigi Health Center IV in the Mpigi district, ≈30 km south of Kampala . His symOn September 26, he began to display hemorrhagic signs, notably profuse bleeding from body orifices. Clinical findings included fever (temperature 38.4°C), nausea, vomiting, diarrhea, musculoskeletal pain, abdominal pain, headache, sore throat, difficulty swallowing, difficulty breathing, anorexia, bleeding from the nose, bloody stools, vomiting blood, and upper gastrointestinal tract bleeding. He died on September 28.Whole blood and serum samples collected on September 28 were sent to the Uganda Virus Research Institute (UVRI)/US Centers for Disease Control and Prevention (CDC) VHF laboratory in Entebbe for testing on September 30. We performed diagnostic testing by real-time reverse transcription PCR (RT-PCR) targeting the MARV NP gene, antigen detection, and MARV IgM ELISA on the suspected sample as described  . On the R. aegyptiacus bats.After laboratory confirmation, a multidisciplinary team from UVRI and CDC-Uganda performed the initial outbreak investigations at Mengo Hospital and Mpigi Health Center IV. The investigation team provided the outbreak case definition, and details of case contacts were obtained from both health facilities. An investigation was also performed in Kasese District, where the health worker was taken for burial. In addition, an ecologic investigation was conducted to identify roosting sites nearby for the potential presence of We created an outbreak database by using the Epi Info Viral Hemorrhagic Fever outbreak management application  and other tropical infectious diseases can confound diagnosis and delay early detection, potentially resulting in large outbreaks that are much harder to control, as was seen during the 2014–2015 Ebola virus outbreak in West Africa  captured in August 2009 in Python Cave, Queen Elizabeth National Park  falls into a cluster that consists mostly of MARV sequences isolated from bats. The closest related sequence was obtained from MARV isolated from a juvenile male nal Park . Other vWe do not know why the patient did not transmit MARV to any of his close contacts. We can assume that none of the contacts had substantial exposure to the patient while he was infectious. The relatively small size of this and previous filovirus outbreaks in Uganda can be attributed to enhanced VHF surveillance, rapid case identification, laboratory testing, and investments from the global health security agenda in rapid sample transportation to the national VHF reference laboratory for diagnostic testing (


We surveyed women in New York, New York, USA, who were in areas with active Zika virus transmission while pregnant. Of 99 women who were US residents, 30 were unaware of the government travel advisory to areas with active Zika virus transmission while pregnant, and 37 were unaware of their pregnancies during travel.  Aedes mosquitoes; the virus can also cross the placenta of infected pregnant women, potentially leading to congenital infection and serious birth defects ; 67 (55.4%) respondents were interviewed in Spanish. We found no statistically significant differences in demographic characteristics between respondents and nonrespondents  Table.Of the 121 respondents, 99 (81.8%) were US residents (considered the United States their home). Approximately one third of the US residents  were unaware of the government advisory (recommending that pregnant women avoid travel to areas with active Zika virus transmission) at the time of travel . Nearly Among the women we surveyed, many were unaware of the government travel advisory, unaware of active Zika virus transmission in areas where they traveled, or unaware of their pregnancy during travel. However, our survey had limitations. The small sample size limited our ability to perform sophisticated analyses, and the potential for social desirability and recall bias are inherent to the study design. The survey questionnaire was not a validated instrument. Also, the women described here completed the survey after Zika virus testing; therefore, it is possible that they had a better understanding than the general public. Most participants in our survey were interviewed in Spanish. This finding underscores the need for providing educational materials in multiple languages. Although our findings cannot be generalized, they provide insight for increased and improved public health messaging. Public health authorities in the United States should continue to raise awareness among women of reproductive age about the risk for Zika virus infection from travel, enabling them to better make informed decisions. Women who are trying to become pregnant or who are pregnant should avoid travel to areas with active Zika virus transmission and, if they must travel, should talk to their healthcare provider first and take steps to minimize exposure to Zika virus. Furthermore, women who are trying to become pregnant should follow Centers for Disease Control and Prevention  guidelines on how long to wait to get pregnant after a potential Zika virus exposure (Additional materials and methods for survey of knowledge about Zika virus infection among pregnant women who were in areas with active virus transmission.


Rotavirus is a common cause of diarrhea worldwide, and vaccination prevents rotaviral gastroenteritis. Since the rotavirus vaccine was introduced in Korea in 2007, the prevalence of rotaviral gastroenteritis has decreased. However, little is known on the economic burden of rotavirus infection and its variations in Korea. Here, we estimated the economic costs of rotavirus infection from 2009 to 2012 using nationwide data from the National Health Insurance Service (NHIS) claims. Socioeconomic costs were subdivided into direct and indirect and measured with a prevalence-based approach. Costs were converted from Won to United States dollars (US$). The number of children <5 years old infected with rotavirus decreased from 21,437 to 10,295 during the study period, representing a decrease in prevalence from 947 to 443 per 100,000. The sum of direct and indirect costs also decreased, from $17.3 million to $9.6 million, and the days of admission decreased from 76,000 to 38,000. However, per capita expenditures slightly increased, from $809 to $934. Thus, the economic burden of rotavirus infection decreased after implementation of rotavirus vaccination. Including the vaccine as part of the national essential vaccination program could reduce the prevalence of and economic loss from rotavirus infection in Korea. Rotavirus (RV) infection is the most frequent cause of diarrhea in young children worldwide . It is eTwo RV vaccines are currently administered to children globally, RotaTeq  and Rotarix  –6. RV5 iAfter the World Health Organization (WHO) recommended the inclusion of the RV vaccine in the national immunization programs (NIPs) of all countries in 2009 , the worIn Korea, the Ministry of Food and Drug Safety approved RV5 in June 2007 and RV1 in January 2008, but RV vaccines were not included in the NIP , 11. Accprevalence-based approach and estimated the costs due to RV infection from 2009 to 2012 in Korea. The prevalence of RV infection is defined as the number of cases of RV infection in the population (obtained from the NHIS claims data) divided by the population by age ) [This study estimated the economic burden of RV infection using a (KOSIS)) . The sta(KOSIS)) .th Revision diagnostic (ICD-10) code A08.0 to identify cases of RV infection used to calculate costs in the NHIS claims database from 2009 to 2012 on the main-disease. Furthermore, to present the sensitivity analysis of prevalence, we estimated RV patients by including the first sub-disease ICD-10 codes and compared this with the main disease in patients.The NHIS was independently established as a single-payer health insurance system in Korea, and the NHIS claims data cover all residents of Korea , 16. We The method used to estimate the economic burden of disease was as follows: Economic Burden = Direct costs + Indirect costs Direct costs included the direct medical care costs  and direct non-medical costs (transportation costs). The NHIS data are nationally representative of medical costs in Korea, so we could calculate the number of patients, admission days, and NHIS-covered medical costs using these data , 19.NHIS-covered service costs included the amount paid by the health insurance and the patients’ copayments, while non-NHIS covered services costs (those not covered by the Korean NHIS) were estimated based on the proportion of uninsured service costs using data from the 2012 Survey on the Benefit Coverage Rate of National Health Insurance for medical charges . This puWe estimated transportation costs using the transportation costs from infectious diseases patients from the Korea Health Panel, which provided data on the patterns of health service use and health expenditures . We consIndirect costs included opportunity costs lost as a result of productivity loss through premature death and morbidity costs. Additionally, caregiver costs were included in the indirect costs. Opportunity costs were estimated via the human capital approach . ProductProductivity loss for inpatient care was calculated according to the average daily wage by sex and age from KOSIS and the period of inpatient hospital days from the NHIS claim data , 13. ProCaregiver costs are the economic losses that result when caregivers stop economic activities to care for patients , 21. ResFor inpatient care, caregiver costs were calculated as the period of inpatient hospital days, and, for outpatient care, the caregiver cost was estimated at about one-third of the daily working hours , 19, 22.All costs were converted into US dollars using the average 2012 exchange rate of the U.S. dollars ($1 = 1126.25 Korean Won) [This study was approved by the Institutional Review Board of Korea University (1040548-KU-IRB-13-164-A-1(E-A-1)(E-A-1)(E-A-1)).The number of patients <5 years of age with RV infection decreased from 21,437 in 2009 to 18,507 in 2010, 16,215 in 2011, and 10,295 in 2012 , Fig 1. The decreasing pattern of RV is also shown when RV cases were included as sub-disease ICD-10 codes .The overall proportion of RV infections occurring in children aged <4 years old was 73.3% in 2009, 73.2% in 2010, 73.7% in 2011, and 76.0% in 2012; thus, >70% of RV infections occurred in children <5 years old . MortaliAccordingly, the total number of days of admission in children <5 years old decreased, from 76,203 in 2009 to 64,736 in 2010, to 63,536 in 2011, and to 37,707 in 2012. The total number of days of admission for all patients also decreased, from 83,599 in 2009 to 44,024 in 2012 . HoweverThe total costs obtained by summing the direct and indirect costs associated with RV infection in children <5 years old increased from $17.3 million in 2009 to $17.9 million in 2010, to $14.6 million in 2011, and to $9.6 million in 2012 , Fig 2A.The direct cost for patients <5 years old decreased from $8.9 million in 2009 to $4.7 million in 2012, or by about half , Fig 2A.Indirect costs for children <5 years old increased from $8.5 million in 2009 to $9.9 million in 2010, but indirect costs in 2011 and 2012 remarkably decreased to less than half of the cost in 2010 ($4.8 million), and the trend related to indirect costs in the overall population was equal to that of children <5 years .Changes in per capita costs were also noted to vary with the economic burden of RV infection. Total costs for patients overall decreased from 2009 to 2012, but per capita of patients <5 years old and in the entire population increased from $808.85, $656.99 in 2009; $966.92, $773.47 in 2010, $901.4 and $770.71 in 2011 and to 933.78, 822.38 in 2012, respectively .Finally, we conducted the sensitivity analysis using the discount rate of cost. When a 3% discount rate was applied, the total costs were 17.6, 16.6, 16.3, and 10.5 million dollars in 2009, 2010, 2011, and 2012, respectively. When a discount rate of 5% was applied, the total cost decreased to 17.3, 15.9, 16.1, and 10.3 respectively .This study estimated the economic burden of RV infection using nationally representative data from the NHIS claims, the Korea Health Panel, and the KOSIS. According to these data, since 2009, the prevalence and admission rates due to RV infection in children have decreased remarkably.RV infection is primarily spread by children <5 years old , and RV In Korea, RV vaccination also appears to be highly efficacious in protecting against RV-AGE. The Korea Centers for Disease Control and Prevention (KCDC) announced that the group A RV detection rate decreased from 12.3% in 2005 to 5.7% in 2013 for viral AGE cases in Korea , 30. AddHowever, the rate of vaccination against RV is still low in Korea. In 2012, the vaccination rate for vaccines included in the NIP, such as BCG and hepatitis B, in 3-year-old children was >90% . At the Although the RV vaccination rate remains low, its effectiveness against RV infection is easily observed , 32. SomThere were 7 fatal cases in Korean children from 2009 to 2012 in the NHIS claims data. Although fatality cases are rather scarce, the burden of RV is substantial. For example, more than 85% of the total number of days of admission were attributable to young children <5 years old. Thus, the indirect costs related to caregiver costs were a great expense to the Korean society, because many women of working-age needed to take care of their ill children as caregivers , 15, 21.Although the number of outpatients and days of admission decreased, per capita expenditures did not; instead, it showed an increasing pattern from 2009 to 2012, hence, the economic costs of the disease remain a burden for each family and the Korean society.With respect to government support for childcare, the number of daycare centers in Korea increased about 8-fold since the 1990s  . There wThe WHO Strategic Advisory Group of Experts (SAGE) on Immunization also recommended adding the RV vaccine to all NIPs, especially in countries in which diarrheal deaths occur in >10% of children <5 years old –38.Since the United Kingdom included the RV vaccine in their national vaccination schedule in July 2013, the rates of attendance and admission for gastroenteritis of any cause reduced by 48% and 53%, respectively in 2014 compared with the mean of cases in the pre-vaccine years 2012 and 2013 . AdditioThis study has some limitations. First, a diagnosis code was used to calculate the number of cases in this study, but RV tests were not performed for all gastroenteritis patients. Thus, there is a difference between the number of patients in our study and the overall number of patients in Korea. Additionally, as gastroenteritis can sometimes be caused by a mixed pathogen , estimatIn conclusion, after RV vaccines were introduced in Korea, the number of patients and the economic effects of RV infection decreased. However, indirect costs with respect to the loss of caregiver’s productivity increased because the infection was mainly found in children aged <5 years and per capita expenses showed an increasing pattern. Therefore, including the RV vaccination in the NIP would contribute to reducing the prevalence of RV infection, economic loss from RV infection, and improving the quality of life in Korean children in the long-term.S1 Table(DOCX)Click here for additional data file.


We studied a clinical case of vaccinia virus that caused an ocular manifestation in a dairy worker in Brazil. Biologic and molecular analyses identified a co-infection with 2 isolates from different Brazilian vaccinia virus phylogenetic groups. The GenBank accession numbers are MG012795  and MG012796 (large).We obtained the complete genomes using the Illumina MiSeq instrument  with the paired-end application. The sequence reads were assembled de novo using ABYSS software (http://www.megasoftware.net). The analysis demonstrated a co-infection with viruses from both VACV-BR groups, such that the large-plaque clone clustered with group 2 VACV-BR isolates and the small-plaque clone clustered with group 1 VACV-BR isolates. We named these isolates Carangola eye virus 1  and Carangola eye virus 2 (large).Analysis of the complete genome revealed 92% similarity between the 2 isolates, and some genes confirm a remarkable variability (Our study demonstrated the genetic and phenotypic variability between 2 viruses isolated from the same sample in a natural human co-infection with VACV. The viruses belong to 2 distinct VACV-BR groups, reinforcing and expanding previous work with other hosts (Ocular vaccinia infection in a 45-year-old male dairy farm worker, Brazil, 2015.