The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and The functionalization of plant virus-based nanoparticles is an emerging field, facilitated by the well-characterized genomic sequence and modular organization of many different plant viruses –3. PlantPotato virus X (PVX) is the type member of the genus Potexvirus (family Alphaflexiviridae) and is often used as a vector for heterologous gene expression in plants [n plants –18. The n plants –21. The n plants , 23. Eacn plants , 24–28. Foot-and-mouth disease virus (FMDV) 2A sequence between the transgene and the cp sequence, yielding a CP fusion protein, free target protein, and wild-type CP due to a ribosomal skip during translation (the overcoat method) [ in vivo tumor imaging in mouse models was recently reported [Genetic modification or chemical conjugation can be used to tailor the surface properties of plant viruses allowing them to be engineered, for example, as drug delivery vehicles, tissue scaffolds, and electronic devices , 29–32.  method) –45. The  method)  or trypt method) , and the method) , 48. A Preported . Arabidopsis thaliana phototropin  2 and can be fused to the movement protein of TMV for quantitative biological imaging applications [Although fluorescent proteins such as mCherry and green fluorescent protein (GFP) are routinely used as reporters for molecular interactions, they are dependent on pH and oxygen, and their large size (at least ~240 amino acids) and complex structure make them more difficult to express –51. The ications –55. The ications , 55, 56.4S-CP (our unpublished data) to create an iLOV-CP fusion via a (Gly4Ser)3 linker (G4S). The integrity of the resulting vectors pPVX-iLOV-CP, pPVX-iLOV-2A-CP, and pPVX-iLOV-G4S-CP was tested by PCR and sequencing. The vectors were propagated in Escherichia coli strain DH5α ready for the inoculation of Nicotiana benthamiana plants as previously described [The PVX-based vectors we used to produce recombinant particles displaying the iLOV polypeptide are shown in escribed . N. benthamiana leaves using the RNeasy Plant Mini Kit  and 3 μg RNA samples were treated with 3 U DNaseI (Thermo Fisher Scientific) as recommended. The corresponding cDNA was produced using M-MLV Reverse Transcriptase RNaseH Minus Point Mutant . We mixed 1 μg of the DNaseI-treated RNA with 0.5 μM of the oligo-dT primer for hybrid PVX particles and 27 μl DEPC-treated water. The mixture was heated to 80°C and then incubated for 10 min on ice to ensure primer annealing before adding 10 μl 5x M-MLV reaction buffer, 8.5 μl DEPC-treated water, 1 mM dNTPs, and 1 μl M-MLV. The cDNA synthesis reaction was carried out for 30 min at 40°C, 20 min at 45°C, 20 min at 50°C, 20 min at 55°C, and 20 min at 70°C. After reverse transcription, residual RNA was degraded with 1 μl 10 mg/ml RNaseA for 1 h at 37°C. PCR was carried out with primers TGB-fw and CX4 and the products were resolved by 1.2% agarose gel electrophoresis to confirm the integrity of the iLOV-CP and iLOV-2A-CP cDNAs.Total RNA was isolated from systemically infectedμl of recombinant PVX cDNA samples with 0.3 μM each of primers CX4i and CX8, 10 μl 2x iQ™ SYBR Green® supermix , and 3.8 μl water. The qPCR program comprised three steps: 3 min at 95°C for polymerase activation and DNA denaturation and then 40 cycles of 15 s at 95°C, 30 s at 55°C, and 45 s at 72°C measured with SYBR Green. Finally, melting curves were constructed from 55°C to 95°C in 0.5°C/2 s steps using primers for the cp (CX4i and CX8) and tgb (CX3i and CX9) as previously described [RNA levels in PVX-infected leaves were determined by quantitative real-time PCR (qPCR). We mixed 5 escribed . Triplic N. benthamiana leaves and were analyzed by SDS-PAGE [Proteins were extracted from systemically infectedSDS-PAGE  and westSDS-PAGE .Systemic infection with recombinant PVX particles was visualized by confocal laser scanning microscopy using a Leica TCS SP instrument . We removed 1 × 1 cm leaf pieces showing fluorescence and mounted them with the abaxial surface facing upwards onto glass microscope slides. An HCX PL APO 63.0 × 5.00 water-corrected objective was used with a drop of water placed directly on the coverslip to image viral cell-to-cell movement across the epidermal cell layer. The excitation and detection wavelengths for iLOV were 476 nm and 510–550 nm, respectively. LCS software was used for image analysis, following import into Photoshop CS5  for contrast adjustments and overlay.Purified iLOV-CP and iLOV-2A-CP virus particles were prepared as described in the protocol published by the International Potato Centre  with modifications as previously described . CentrifImmunosorbent electron microscopy (ISEM) was carried out with virus-containing plant extracts from systemically infected leaves or purified recombinant viruses. Pioloform-coated nickel grids  were incubated on a drop of monoclonal antibody MAC58 against PVX  for 20 min at room temperature to capture the recombinant PVX particles. Immunogold decoration of captured or adsorbed particles with primary anti-PVX antibody or the anti-iLOV antiserum and secondary goat anti-rabbit (GAR) 15 nm gold conjugate  and imaging was carried out as previously described . cp gene using different strategies: (a) a direct fusion, (b) via a (Gly4Ser)3 linker, and (c) via the FMDV 2A sequence  and systemic infection was observed 6 dpi, as revealed by the general green fluorescence. These plants also displayed more severe symptoms than typically observed for systemic infection with wild-type PVX, including mosaic patterns and dwarfing, and necrotic lesions appearing from 10 dpi onwards  and cp genes  and subgenomic RNAs (sgRNAs) 1 and 2 but not sgRNAs 3. This allowed a direct correlation between the iLOV-(2A)-CP RNA level and the total amount of PVX RNA. The quantity of tgbp3 and cp RNA was correlated to PP2A mRNA and the relative amount of cp to tgb3 was calculated and compared. We observed higher relative cp RNA levels for the iLOV-2A-CP fusion than for the iLOV-CP direct fusion  was carried out to determine the levels ofa plants . Total Rcp genes . We thent fusion . This waExtracts were prepared from leaves containing the pPVX-iLOV-CP and pPVX-iLOV-2A-CP fusion proteins for analysis by SDS-PAGE and western blot. The iLOV-CP and iLOV-2A-CP fusion proteins were detected with the anti-PVX antibody and the anti-iLOV antiserum with molecular weights of 38.0 and 39.9 kDa, respectively, as expected Figures . HoweverThe ability of the fusion proteins to assemble into PVX particles was confirmed by electron microscopy. PVX particles were immunocaptured from leaf extracts using monoclonal antibody MAC58, detected with a primary anti-PVX antibody and decorated with GAR 15 nm gold conjugates Figures . TypicalTo gain insight into the cell-to-cell movement of hybrid PVX particles, leaves systemically infected with pPVX-iLOV-CP and pPVX-iLOV-2A-CP were studied by confocal laser scanning microscopy . The iLOPVX particles displaying iLOV were purified with yields of 136–206 mg per kg of infected leaf material by adapting the purification protocol for wild-type PVX particles developed by the International Potato Centre. The different pooled fractions were tested for the presence of iLOV-CP and iLOV-2A-CP by western blot . Both th4S-CP) was not, most likely due to size limitation [4S-linker enables iLOV to move between the CP subunits and prevents particle assembly, we replaced the G4S linker with the more rigid AK3A linker (three copies of the sequence AKKKA). The plants showed neither green fluorescence in inoculated leaves nor systemic infection at 28 dpi (data not shown).The biological compatibility and multivalency of genetically and chemically addressable sites on virus CPs have resulted in a broad range of applications for plant virus nanoparticles. In this study, PVX-derived vectors  were genmitation , 64, 65,Systemic infection with pPVX-iLOV-CP was delayed and the infection symptoms were mild, whereas rapid systemic infection was established following inoculation with pPVX-iLOV-2A-CP and the infection symptoms were severe, including necrosis Figures . This ma Tobacco mosaic virus (TMV) [ Plum pox virus [ Cowpea mosaic virus [When certain peptides or proteins are fused to plant virus CPs, the assembly of the virion is sterically or electrostatically inhibited and systemic movement prevented , 64, 65.ox virus , and 50 ic virus . With thic virus , whereasic virus . Fusion ic virus  as shownic virus . The ratic virus , 71, alt cp RNA level in relation to the tgbp3 RNA by qPCR because the PVX tgbp3 sequence is found on the gRNA and all sgRNAs except that carrying the cp gene, whereas the cp sequence is found on the gRNA and all sgRNAs. The comparison of cp and tgbp3 RNA levels therefore provides an estimation of cp RNA levels with a built-in internal control. The level of iLOV-CP RNA was lower than that of iLOV-2A-CP RNA (We investigated theA-CP RNA , explain cp sequence at 34 dpi as reported for iLOV fusions to the TMV CP [Some PVX coat proteins with N-terminal extension peptides cannot form particles due to steric inhibition , the nume TMV CP . However cp gene [The presence of iLOV-decorated PVX particles in systemically infected leaves was confirmed by western blots and transmission electron microscopy Figures  and 5. W cp gene . These rConfocal laser scanning microscopy revealed that the recombinant iLOV-CP and iLOV-2A-CP particles were potentially able to pass between epidermal cells, as indicated by the presence of a fluorescent signal in the plasmodesmata . SimilarFluorescent proteins can provide insight into many cellular processes and interactions but also suffer from drawbacks such as their complex structure, large size, pH, and oxygen dependence , 51. TheWe have demonstrated that the 113-amino-acid fluorescent protein iLOV can be directly fused to the N-terminus of the PVX coat protein without the help of a flexible linker and is displayed on the particle surface. Because the iLOV protein has several advantages over other fluorescent proteins, it will be useful not only for the analysis of VRCs and long-distance virus movement, which currently relies on the production of overcoat structures, but also as a new platform for the molecular imaging of tumors. We recently reported an mCherry PVX overcoat system for tumor imaging in mouse models . Our PVX


A virus that caused blisters and ulcers in pigs in China was detected to be a strain of Senecavirus A (SVA). Complete genome sequencing and analysis results showed that the isolate shares 93.8% to 99.1% identity with other isolates that have been reported, proving that a new isolate of SVA was found in China. Senecavirus and family Picornaviridae and is closely related to members of the genus Cardiovirus. It is a nonenveloped, single-stranded, positive-sense RNA virus (Senecavirus A (SVA) belongs to the genus NA virus . PigletsNA virus . The virNA virus  but was NA virus , 5. The NA virus .In March 2016, a similar outbreak causing blisters and ulcers occurred on a swine farm in south China. Samples were collected, and a one-step reverse transcription-PCR kit  was used to identify the pathogen. The result showed that it was a strain of SVA and not a strain of foot-and-mouth disease virus, swine vesicular disease virus, or vesicular stomatitis virus. This strain was named SVV HN16 (GenBank accession no. MF893200). To study the genomics of this SVA isolate, the complete genome was amplified by means of segmented cloning. Then, the target fragments were purified, inserted into pMD19-T vectors, and sequenced by Ruibio Biotech in Beijing, China.The sequencing results suggested that isolate SVV HN16 consisted of 7,280 bp, in which the 5′ untranslated region (UTR) contained 668 bp and the 3′ UTR, excluding the polyA tail, contained 66 bp. Moreover, an open reading frame contained 6,546 bp, encoding a polyprotein with 2,181 amino acids. Homology analysis with the DNASTAR Lasergene 7 package found that the homology between this strain and other isolates from Thailand (GenBank accession no. KY368743 and KY368744), Canada (KY486161 and KY486166), China (KX173338 and KT321458), Brazil (KR063107 and KR063108), and the United States (DQ641257 and KT757280) was between 93.8% and 99.1%; the homology of the polyproteins was between 97.8% and 99.8%, confirming that a new isolate of SVA was found in China.MF893200.The whole-genome sequence of SVA strain SVV HN16 has been deposited in GenBank under the accession number


The presence of the Zika virus (ZIKV) infection has gone ahead to be a threat to people based on its adverse impacts. More specifically, the pregnant women have been discouraged from traveling to the areas affected by the ZIKV because of the likelihood of the virus causing congenital abnormalities especially the microcephaly. The pregnant women probably attracted the virus during their first trimester while visiting ZIKV affected territories. Although the ZIKV infected cases have reduced in some parts of countries, the global risk assessment has not been changed. The virus continues to spread geographically to areas where competent vectors are present. At present, there is still no treatment of ZIKV related illness, especially microcephaly. The Zika virus (ZIKV) was discovered during a study conducted in 1947 aimed at establishing the causative agents of yellow fever, dengue, and other West Niles viruses . The virThese include Aruba, Barbados, Bolivia, Brazil, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Guatemala, Haiti, Panama, Trinidad and Tobago, and American Samoa among others . TherefoAedes mosquitoes . Some women had reported rashes that were seen on the faces, palms and soles. However, several studies have also noted that not all these symptoms present in women were infected by the ZIKV. Some of the cases tested showed that some women who were infected had no physical signs . Biologically, the growth and development of the brain happen concurrently with the head. The condition occurs when there is an inadequate development of the brain during the prenatal stage in the head and causing the brain to stop growing after childbirth. The condition can occur in isolation or occur together with other congenital defects prevalent among the fetuses and babies who have been infected by the virus during pregnancy (Zika virus has been linked to some of the birth defects experienced by mothers who were infected with the virus during their pregnancy . The mosregnancy . Severe regnancy . Childreregnancy . They alregnancy . Congeniregnancy . An infaregnancy . The conFurthermore, results from the studies published in the New England Journal Medicine discovered more relationships between the ZIKV infection and the causes of microcephaly and brain anomalies in newborn babies . The assAedes mosquitoes.The other studies reported also examined the possible link between ZIKV infection and congenital birth defect in the case of microcephaly, which is a common type of brain abnormality believed to be caused by the ZIKV . The repIn this review, an effort was made to unfold a close link between ZIKV infection during pregnancy and congenital abnormalities. The first confirmed evidence of this association is based on the Pan American Health Organization and World Health Organization reports which updated on the status of the ZIKV infection based on the discoveries made in Brazil and French Polynesia in 2015 and 2014, respectively. However, these studies were never based on direct factual proofs, but relied on fetal malformations which were associated with the infection of ZIKV . The relIR wrote the initial draft, JL and VB designed the manuscript, Y-HP did critical analysis and approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepadnaviridae, Polyomaviridae, Poxviridae, Papillomaviridae and Adenoviridae, all of which possess double-stranded DNA genomes, exhibited more frequent co-divergence than the other virus families studied here. At the other extreme, the virus and host trees for all the RNA viruses studied here, particularly the Rhabdoviridae and the Picornaviridae, displayed high levels of topological incongruence, indicative of frequent host switching. Overall, we show that cross-species transmission plays a major role in virus evolution, with all the virus families studied here having the potential to jump host species, and that increased sampling will likely reveal more instances of host jumping.The cross-species transmission of viruses from one host species to another is responsible for the majority of emerging infections. However, it is unclear whether some virus families have a greater propensity to jump host species than others. If related viruses have an evolutionary history of co-divergence with their hosts there should be evidence of topological similarities between the virus and host phylogenetic trees, whereas host jumping generates incongruent tree topologies. By analyzing co-phylogenetic processes in 19 virus families and their eukaryotic hosts we provide a quantitative and comparative estimate of the relative frequency of virus-host co-divergence versus cross-species transmission among virus families. Notably, our analysis reveals that cross-species transmission is a near universal feature of the viruses analyzed here, with virus-host co-divergence occurring less frequently and always on a subset of viruses. Despite the overall high topological incongruence among virus and host phylogenies, the  Emerging infectious diseases are often characterized by host switching events, in which a pathogen jumps from its original host to infect a novel species. However, given the ecological and genetic barriers a virus must overcome to jump species and adapt to new hosts, it might be reasonable to assume that successful cross-species transmission is a relatively rare occurrence and that viruses are instead more likely to co-diverge with their hosts. Using a comparative co-phylogenetic analysis performed at the scale of virus family we have revealed that co-divergence is relatively infrequent among 19 diverse families of RNA and DNA viruses, such that cross-species transmission plays a central role in virus evolution. Host jumping was especially common in viruses with RNA genomes, and by drawing broad-scale comparisons our analysis reveals which virus families have a greater propensity to jump species barriers and hence successfully emerge in new hosts. Finally, our data suggest that sampling more viruses increases the likelihood of detecting host jumping events. Emerging pathogens that cross the species barrier to infect new hosts can profoundly affect human and animal health, as well as wildlife and the agricultural industries. Although most emerging diseases seemingly result from such a process of cross-species transmission, it is also the case that some viruses seem to rarely jump the species barrier and instead co-diverge with their hosts over long stretches of evolutionary time. For example, long-term virus-host co-divergence has been suggested to play a key role in the evolution of vertebrate herpesviruses over periods of ~400 million years  and inseGiven the evolutionary and ecological barriers a virus must overcome to cross the species barrier and successfully establish itself in a new host, it might seem reasonable to assume that successful cross-species transmission is a relatively rare occurrence . Indeed,Clearly, identifying the relative frequencies of co-divergence versus cross-species transmission is of central importance to understanding the basic mechanisms of virus evolution and disease emergence. In particular, it is important to determine whether some virus families have a greater propensity to jump hosts than others and, if so, what factors govern this pattern. Currently, however, there is no quantitative or comparative measure of the frequency of cross-species transmission versus co-divergence, so that determining whether one virus family is more likely to jump species boundaries than another is difficult to assess. One simple and powerful way to estimate these key evolutionary parameters is via ‘co-phylogenetic’ analysis that assesses the degree of phylogenetic congruence (i.e. similarity) between hosts and their parasites . In partTo date, co-phylogenetic studies of viruses have largely focused on the evolution of a subset of viruses within a particular virus family, and have not been performed in a comparative manner. For example, although there has been much work dedicated toward describing co-divergence in herpesviruses, these studies generally only encompass one particular host type (e.g. primates ) and so To provide a quantitative measure of host switching we compared 19 virus families, incorporating viruses infecting a diverse sample of eukaryotic hosts including mammals, birds, reptiles, amphibians, fish, plants and insects. Under the measure we utilize here, when nPH85 = 0 between the virus and host trees it implies that their topologies are identical such that there is very strong evidence for co-divergence . ConversOur analysis considered a total of seven DNA and 12 RNA virus data sets that provided sufficient data to perform a quantitative co-phylogenetic analysis. Hence, the study relied heavily on specific selection criteria see  that necTo determine the prevalence of host switching between different viruses, we inferred family-level viral phylogenies and compared these to phylogenies of their hosts. Importantly, our analytical approach—which utilizes the nPH85 distance—provides a relative measure of phylogenetic congruence that is directly comparable between data sets that differ in size (i.e. different number of viruses and host species). Our method assumes that viruses that have co-diverged with their hosts will share the same tree topology. In contrast, an increasing number of host jumping events should lead to greater phylogenetic incongruence. The reasoning behind this assumption is that there exists a very large number of possible phylogenetic tree topologies even for data sets with a few samples, such that similarities between a pair of virus-host trees (i.e. congruence) are highly unlikely to arise by chance. Of course, phylogenetic events other than cross-species transmission might also lead to phylogenetic incongruence and we test the validity of this assumption later in the manuscript.Hepadnaviridae (double-stranded DNA) had the shortest distance (nPH85 = 0.6), indicating that this family experiences more frequent co-divergence than any other studied here. At the other end of the spectrum both the Rhabdoviridae and Picornaviridae (single-stranded RNA) displayed nPH85 > 0.97, indicative of frequent host switching and hence little evidence for virus-host co-divergence.Across the data set as a whole we found that all virus families displayed relatively large tree topological distances with nPH85 values of ≥0.6, suggesting that cross-species transmission is widespread, at least at the family-level . While aWe also investigated when the species jumping events occurred in the evolutionary history of the virus families. To do this, we determined whether phylogenetic incongruences tended to occur in deeper sections of the phylogeny or to more shallow nodes in the tree. Accordingly, we considered the number of nodes subtending clades in the host tree that are not present in the virus tree, a metric known as ‘node depth’. Nodes that are deep correspond to clades that are more diverse, and often older, than those clades subtended by shallower nodes. For each pair of virus-host trees we calculated the depth of every node that differed within each virus-host pair and divide each depth by the maximum node depth . This noHepadnaviridae and Poxviridae exhibit some clear matches with the evolutionary histories of their respective hosts. Most notably, their co-phylogenies show a clear segregation between distinct clades that are associated with a specific host type . Conversely, the phylogenies of most RNA viruses appear to largely mismatch those of their hosts.Tanglegrams depicting pairs of rooted phylogenetic trees display the evolutionary relationship between each virus family and their host species . DespiteHepadnaviridae–see below), and lineage duplication and extinction playing a much more minor role. We next reconstructed the history of these evolutionary events in detail in the Hepadnaviridae (i.e. the most co-divergent virus family). This revealed that under the most likely co-phylogenetic scenario the proportion of cross-species transmission represents 0.57 of all events  and five (i.e. Togoviridae) host types. Notably, we found a significant association between the number of viruses per virus family and the nPH85 (p<0.005)  . Importap<0.005) . Note thUnderstanding how viruses and their hosts co-evolve is central to revealing the nature of virus evolution and the determinants of disease emergence. In particular, we lack a quantitative understanding of whether some types of virus, such as those classified into different families or that possess genomes of different nucleic acid types, are better able to jump species boundaries compared to others. To investigate the comparative prevalence of cross-species transmission among viruses we measured the congruence between virus and host phylogenetic trees using a normalized tree topological distance-based approach . If taxHepadnaviridae, Polyomaviridae, Poxviridae, Papillomaviridae and Adenoviridae had the shortest nPH85 distances and were thus relatively more host-specific than the other virus families analyzed here. This is supportive of earlier suggestions that some DNA viruses have a long history of co-divergence with their hosts [Herpesviridae [Poxviridae [Polyomaviridae [Flaviviridae that infect primates—the pegiviruses and hepaciviruses, [Despite the overall large nPH85 distances observed among all virus families, our data also revealed that the ir hosts , which isviridae , the Poxxviridae  and the aviridae . Howeveraviridae  ′ corresponds to the clades that are not shared between the pair (i.e. those that are unique to each tree). The actual PH85 distance is twice the number of unique clades. To normalize this metric we divide PH85 by the maximum distance by considering the two tree topologies, randomizing the tips for one of the trees 1000 times, and calculating PH85 for each replicate  by comparing, in a quantitative manner, the tree topologies for viruses and their corresponding hosts. To this end we calculated a normalized PH85 tree topological distance , referreTo determine whether host jumping occurred more often toward the root or tips of the trees, we calculated the relative node depth for incongruent nodes between virus-host pairs of trees see . This meHepadnaviridae in more detail as this family contains the best evidence for co-divergence Click here for additional data file.S2 Fign randomizations.The red, dashed line illustrates the PH85 distance of the non-randomized data, while the black, solid line is the PH85 distance after randomizing the data after (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableVirus genera were excluded either due to lack of available data or because we were unable to obtain a reliable alignment of sufficient length for phylogenetic analysis (i.e. at least 100 amino acids after trimAl pruning).(DOCX)Click here for additional data file.S3 TableThe overall nPH85 distances are illustrated in (DOCX)Click here for additional data file.


We found substantial virus prevalence (25.8–35.7%) in all ecosystems, but prevalence and identified family-level virus diversity were greatest in cultivated areas, with some virus families displaying strong agricultural associations. Our survey revealed 94 previously unknown virus species, primarily from uncultivated plants. This is the first effort to systematically evaluate plant-associated viromes across broad agro-ecological interfaces. Our findings indicate that agriculture substantially influences plant virus distributions and highlight the extent of current ignorance about the diversity and roles of viruses in nature.Disease emergence events regularly result from human activities such as agriculture, which frequently brings large populations of genetically uniform hosts into contact with potential pathogens. Although viruses cause nearly 50% of emerging plant diseases, there is little systematic information about virus distribution across agro-ecological interfaces and large gaps in understanding of virus diversity in nature. Here we applied a novel landscape-scale geometagenomics approach to examine relationships between agricultural land use and distributions of plant-associated viruses in two Mediterranean-climate biodiversity hotspots (Western Cape region of South Africa and Rhône river delta region of France). In total, we analysed 1725 geo-referenced plant samples collected over two years from 4.5 × 4.5 km Over the next 30 years the world’s human population is expected to increase by 33%, reaching 9.7 billion by 2050 . IncreasAlthough almost 50% of the microbes responsible for emerging plant diseases are viruses . For exaTo develop mechanistic understanding of how viral pathogens might emerge in crops, it is essential to identify the parameters that determine viral diversity and prevalence across agro-ecological interfaces. Initial work in other situations indicates, for example, that reductions in plant diversity can increase prevalence of plant pathogens, including some viruses  virus prevalence is greater in cultivated areas in both locations, but that plant diversity is negatively associated with family-level diversity of plant-associated viruses, contrary to expectations; (2) some virus families show strong associations with agriculture; and (3) the novel viruses identified (94 putative species) are primarily from uncultivated plants.2 sampling grids across agro-ecological interfaces in two different Mediterranean-climate areas: the Rhône river delta in Southern France and the Cape Floristic Region in South Africa. Both regions represent unique vegetation and have been designated as UNESCO World Heritage Sites  of 2010 and 2012. We navigated to each geo-node with a Trimble Geo XT V6 (50 cm precision). At each visit, we photographed the geo-node area, rated land use conditions, and collected 5 g of leaf and stem tissue from each dominant plant species for virus analysis. Land use types were rated on a five-point scale: (0) intact native communities; (1) native communities degraded by disturbance or invasion; (2) fallow and old fields; (3) low-intensity polyculture (woodlots and pasture); and (4) intensive crop monoculture. Types 0–1 were considered to be uncultivated , and types 2–4, cultivated .Poaceae); for these, it was necessary to sample from multiple (2–10) individuals to reach the required tissue quantity. In crop monocultures, in which there was little other vegetation, we collected three separate 5-g samples of the crop species , filtered through a 0.45 μm sterile syringe filter and centrifuged at 148000g for 2.5 h at 4 °C to concentrate viral particles. Unencapsidated nucleic acids were then eliminated by DNase I and RNase A . Total nucleic acids were extracted as a mixed RNA/DNA solution from resuspended virus-particles using a NucleoSpin 96 Virus Core Kit . In total 23 samples were extracted in each VANA batch along with a dual extraction control: sugarcane tissue infected with a unique, known sugarcane bacilliform badnavirus . This dual control served both as a positive with known sequence (the badnavirus) and a negative for the detection of potential contamination (any virus other than the known badnavirus). Before field samples were extracted, sensitivity tests were conducted with 62 different viruses from eleven viral families with a range of genomic nucleic acid types (RNA and DNA), in both single and co-infections; this unique collection of test viruses was available in vivo from the CIRAD quarantine station collection  synthesis was performed on extracted RNA/DNA solutions using the primer, DoDec (5′-CCT TCG GAT CCT CCN NNN NNN NNN NN-3′). Additional controls of nucleic acid-free water (blanks) were added to each sample group at the reverse transcription step and carried through to sequencing. Priming and extension were then performed with Large (Klenow) Fragment DNA polymerase  on all cDNA and genomic DNA in each tube. Next, PCR amplification was carried out using one of the 96 multiplex identifier (MID) tagged primers listed in agrep  to identify open reading frames that were >20 codons long. For each potential open reading frames, a protein sequence translation was aligned using MUSCLE 3.7  or viruses infecting plant-associated fungi . We refer to these plant viruses and mycoviruses collectively as plant-associated viruses (PLAV). Initial classifications may be revised in the future based on further characterization and mycoviruses that are here classified as viruses that probably infect fungi may in fact infect plant hosts and be re-classified as plant viruses. About half of known partitiviruses infect plants, while others infect fungi (BlastN or BlastX) and phylogenetic analyses did not indicate that these reads could be confidently assigned to either one of the two main plant-infecting partitivirus clades  were individual samples. Bulked samples of smaller-statured species were 94% Blast matches, and did not attempt to enumerate genera or species. We adopted this conservative approach because we wanted to avoid ‘over-counting’ different reads or contigs from recombinant viruses as if these were an indication of two separate viruses (a co-infection). To estimate virus diversity at each geo-node, we next calculated the Shannon–Wiener index (H), where species richness (s) was conservatively estimated by virus family richness and the proportion of individuals belonging to each virus family i (pi) was estimated as the number of samples at that geo-node in which the family was represented. Mann–Whitney U-tests were used to evaluate differences between cultivated areas and uncultivated areas with respect to viral prevalence and diversity.To evaluate virus diversity, we first enumerated virus family richness per plant sample. In each sample, we counted only the number of virus families represented (if any), as determined by p-values for multiple testing. All analyses were processed using the VEGAN .To evaluate potential associations between land use and the identity of virus families at geo-nodes, we used RLQ analysis with the fourth corner method . With thhe VEGAN  and ade4BlastN and BlastX determination of sequence similarity with detailed expert review of each taxon identified. On the basis of pairwise sequence similarity, related groups of virus-like sequences  were tentatively assigned to known plant virus families. We then generated maximum likelihood phylogenetic trees from alignments of OTU protein sequences with homologous GenBank accessions using PhyML 3.1 implemented in MEGA version 6.06 , Chrysoviridae (57 out of 439), and Totiviridae (88 out of 439). An additional 128 reads/contigs appeared to represent single stranded DNA (ssDNA) viruses that might infect fungi  and South Africa (2010), using VANA semi-purification and 454 pyrosequencing , and detct fungi .In sensitivity tests where we analysed plants with known virus infections, test viruses were successfully detected in 75.9% (104 out of 137) of cases. As 7 out of 40 of the test viruses were detected by only a single VANA-read  in cultivated plants relative to uncultivated plants  in cultivated areas than it was in uncultivated areas  in France and South Africa. These analyses indicated that, despite some variability over time, the members of several virus families tended to be found significantly more frequently in cultivated areas than in uncultivated areas . In FranPoaceae crops (rice and wheat) there were experiencing recurrent infection with these virus families, including with viruses such as Barley yellow dwarf virus (BYDV) and Barley stripe mosaic virus (BSMV). Were agricultural pathogens like these persisting between crop cycles within nearby uncultivated plants, and/or spilling over from crops into uncultivated (non-crop) hosts? We found OTUs closely related to 18 known crop pathogens in 37 uncultivated plant hosts (59.5% of which were located in uncultivated areas) at the French site and 10 (20% in uncultivated areas) at the South African site  and phylogenetic analyses to assign related groups of virus-like sequences (OTUs) to known plant virus families . This suggests that even in well-studied cultivated host species, there likely remain large numbers of undiscovered plant viruses.Are viruses found in uncultivated hosts likely to be more dissimilar to known viruses than are viruses detected in cultivated hosts , 2012? WOur findings reveal the breadth and abundance of plant-infecting viruses in agro-ecological landscapes, where infection is found throughout cultivated and uncultivated plant communities alike. In matched surveys in French and South African sites, we found more than 120 plant virus OTUs representing 19 of the 22 currently recognised plant-infecting virus families. Ninety-four of these OTUs likely represent novel virus species or genera, with potential, when fully characterized, to enlarge the list of known plant virus species by as much as 7.2%. Besides confirming that currently known plant-infecting virus species are likely a tiny fraction of the total occurring in terrestrial environments, our spatially-informed metagenomics-based approach has provided the most convincing evidence yet of the impact of agriculture on the distribution, prevalence and diversity of plant viruses in the environment. It remains to be determined whether preferential associations of specific virus groups with ecologically disturbed areas, or increased plant virus prevalence within such areas, increase the probability of pathogen emergence.


Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016’ by K Steinhagen et al., the title of the y-axis in Figure 2B was corrected to read ‘Anti-Zika virus IgG ELISA (ratio)’ on 22 December 2016.In the article ‘


Several orthobunyaviruses are important arthropod-borne pathogens, responsible for a variety of diseases in humans, from acute febrile illness to encephalitis.We collected serum samples from a series of dengue suspected cases in Tefé, a mid-size city located in the interior of the Amazonas state, Brazil. Viral RNA extraction was performed, and specimens were tested for dengue virus using RT-PCR. Thirty dengue negative samples were further tested for Mayaro virus (MAYV) and Oropouche virus (OROV) using an RT-qPCR protocol previously described. Positive samples were characterized by MegaBLAST analysis over the entire nucleotide collection of the main public databases, and also by maximum likelihood phylogenetic reconstruction of the S genome segment.We detected nine OROV or OROV-like positive cases among 30 patients reporting fever and headache, as the most common symptoms. The closest nucleotide sequence returned from the MegaBLAST analysis belongs to an OROV isolated in Peru 2008. Moreover, all Tefé samples grouped in the same clade with the OROV reference sequence and other closely-related OROV-like viruses.Dengue viruses are still the most important arbovirus worldwide, causing hundreds of millions of infections every year. Nonetheless, other arboviruses like chikungunya virus, Zika virus, and yellow fever virus have emerged in the last few years and are now a public health concern in several countries. OROV is believed to have caused more than 500,000 febrile infections in Brazil over recent decades. Therefore, the results described in this study strengthen that this arbovirus, and its closely-related recombinants, should be under continuous surveillance, at least in the endemic countries of Latin America. Between April and June 2015, professionals of the health surveillance system at Tefé , a mid-size city located in the interior of the Amazonas state, Brazil, collected serum samples from a series of dengue suspected cases. The specimens were obtained between the first and second day after symptom onset and sent to Fundação de Medicina Tropical – Dr. Heitor Vieira Dourado (FMT-HVD) at Manaus, Amazonas, Brazil for dengue virus (DENV) testing using RT-PCR ,3,4,Peribunyaviridae family, genus Orthobunyavirus), targeting the S segment; as well as for Mayaro virus  aiming the NSP1 coding region Thirty DENV negative samples were randomly selected and sent to Instituto Leônidas e Maria Deane (ILMD), Fiocruz Amazônia, to investigate other Amazonian endemic arboviruses 2We detected nine OROV or OROV-like positive cases , seven males and two females, with a mean age of 36.2 years (range 8 - 63), living in either rural or urban areas of Tefé. Regarding the clinical symptoms, all nine patients reported fever and headache; two patients also reported myalgia and arthralgia and one showed retro-orbital pain. Therefore, these patients presented non-specific symptoms that could easily lead to dengue misdiagnosis, if no specific laboratory testing were applied.To further confirm these results, all nine samples were submitted to an RT-PCR protocol targeting the S segment of OROV/OROV-like viruses using primers OROV_S_134F 5'- CGGACAAGTGCTCAATGCTG -3' and OROV_S_734F 5'- GTCAATTCCGAATTGGCGCA - 3', generating a 601bp amplicon. These products were used for capillary nucleotide sequencing in an ABI3130 genetic analyzer, installed at the ILMD genomics platform. After removal of the primers, the final 558bp sequence for each sample was used for a multiple alignment with MAFFTWe also conducted a BLAST analysis  Bunyavirales order. This new order contains eight new viral families  and one family that was renamed: Peribunyaviridae, formerly Bunyaviridae. Two genera are included in the Peribunyaviridae family, genus Herbevirus and genus Orthobunyavirus Recently, the International Committee on Taxonomy of Viruses (ICTV) made several changes in virus taxonomy, comprising the creation of theOrthobunyavirus genus . According to the ICTV Virus Metadata Repository , among those 48 orthobunyaviruses species, 35 have sequence data deposited in GenBank regarding the S segment. Thus, we used the PhyML software http://tree.bio.ed.ac.uk/software/figtree/.Currently, ICTV recognizes 48 species in the Oropouche orthobunyavirus (RefSeq NC_005777) and the closely-related OROV-like viruses . One exception was observed for the Facey’s Paddock virus, another OROV-like virus isolated initially in Australia 1974, which grouped with the Manzanilla orthobunyavirus isolated in Vietnam, 2004  is less well studied but is believed to have caused more than 500,000 febrile infections in Brazil over recent decades 15FGN is funded by Fundação de Amparo à Pesquisa do Estado do Amazonas - FAPEAM ; Conselho Nacional de Desenvolvimento Científico e Tecnológico  and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior  call MCTIC/FNDCT -CNPq / MEC-CAPES/ MS-Decit 14/2016 - Prevenção e Combate ao vírus Zika. RMPF is funded by Fundação de Amparo à Pesquisa do Estado do Amazonas - FAPEAM . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors have declared that no competing interests exist.All partial S segment orthobunyavirus sequences are available as a FASTA file at https://doi.org/10.6084/m9.figshare.5458342.v1/.Dr. Felipe Gomes Naveca (felipe.naveca@fiocruz.br) and Dr. Regina Maria Pinto de Figueiredo (figueiredormp@yahoo.com.br).


Due to the limited coding capacity of viral genomes, plant viruses depend extensively on the host cell machinery to support the viral life cycle and, thereby, interact with a large number of host proteins during infection. Within this context, as plant viruses do not harbor translation-required components, they have developed several strategies to subvert the host protein synthesis machinery to produce rapidly and efficiently the viral proteins. As a countermeasure against infection, plants have evolved defense mechanisms that impair viral infections. Among them, the host-mediated translational suppression has been characterized as an efficient mean to restrict infection. To specifically suppress translation of viral mRNAs, plants can deploy susceptible recessive resistance genes, which encode translation initiation factors from the eIF4E and eIF4G family and are required for viral mRNA translation and multiplication. Additionally, recent evidence has demonstrated that, alternatively to the cleavage of viral RNA targets, host cells can suppress viral protein translation to silence viral RNA. Finally, a novel strategy of plant antiviral defense based on suppression of host global translation, which is mediated by the transmembrane immune receptor NIK1 (nuclear shuttle protein (NSP)-Interacting Kinase1), is discussed in this review. Due to their sessile nature, plants are constantly exposed to extreme adverse conditions that affect negatively their growth and development, thereby resulting in considerable yield losses worldwide. Among the biotic factors, virus infections are one of the most prevalent agricultural constraints as they often suppress the plant defenses and severely limit productivity of relevant crops, representing a serious threat to global food security. As obligatory intracellular parasites, plant viruses depend on the host cell machinery to replicate their genome, express their genes and invade their hosts. Thus, in order to establish a productive infection, compatible interactions between viral and host factors must occur, from the expression and replication of the viral genome until the cell-to-cell movement and long distance translocation through the vascular system of viral particles. In addition to providing basic compatibility, interactions between viral and host proteins are necessary to modulate the viral infection, preventing or neutralizing the plant defense mechanisms.2+ ions from the extracellular space and/or anion flux, MAPK-mediated signaling, production of reactive oxygen species (ROS), salicylic acid (SA) accumulation, extensive transcriptional reprogramming and activation of defense responses  has recently emerged as a new paradigm of antiviral defenses in plants. In this case, the activation of the transmembrane immune receptor NIK1 [nuclear shuttle protein (NSP)-interacting kinase 1] promotes the down-regulation of translational machinery-associated genes, culminating in the inhibition of viral and host mRNAs translation, which causes an increase in tolerance to begomoviruses (Pea seed-borne mosaic virus (PsbMV) and cyv2 against Clover yellow vein virus (ClYVV) in pea; mo1(1), mo1(2) in lettuce against Lettuce mosaic virus (LMV); pvr1, pvr2 and pvr6 in pepper against Tobacco etch virus (TEV), Potato virus Y (PVY), Pepper veinal mottle virus (PVMV); rym4 and rym5 in barley against Barley yellow mosaic virus (BaYMV); nsv in melon against Melon necrotic spot virus (MNSV); pot-1 in tomato against PVY and TEV; lsp1 in Arabidopsis against Turnip mosaic virus (TuMV) and TEV (Rice yellow mottle virus (RYMV) and Rice tungro spherical virus (RTSV) in rice (Cucumber mosaic virus (CMV) multiplication and Turnip crinkle virus (TCV) in Arabidopsis  binds directly to eIF(iso)4G, rather than to eIF4E isoforms  from several potyviruses as an interacting partner of the translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E in yeast two-hybrid and g assays   has been demonstrated to depend on eIF(iso)4E for its systemic spread in Arabidopsis, suggesting a role of eIF(iso)4E in viral movement (eIF(iso)4E gene did not affect global cellular or viral translation and, in the null mutant, viral replication was still observed. These results indicate that, for the TEV-Arabidopsis system, eIF(iso)4E is not required for viral translation and replication in the viral infective cycle, but is required for viral movement, as TEV systemic spread was completely abolished in the eif(iso)4f knockout lines.Although the VPg-eIF4E/iso4E complex may support viral RNA translation in some potyvirus-host interactions, it probably contributes to other steps of the infection. Accordingly, Turnip yellow mosaic virus (TYMV) possesses a tRNA-like structure at 3’ UTR instead of a poly(A) tail, which works as a translation enhancer of the viral RNA. This region interacts with the host factor eEF1A and seems to regulate viral replication (Potato virus A (PVA) infection (atrh8 mutant plants were resistant to both plant potyviruses Plum pox virus (PPV) and TuMV.Other components of the host translation machinery, such as eEF1A and PABP, are found in the virus translation/replication complex . eEF1A, lication . The ribnfection . These pAdditionally to their roles in the host translation of mRNAs, translation factors play other biological functions that might be exploited by viruses. eIF4E has been shown to accumulate in nuclear bodies, where it is involved in the export of a subset of mRNAs containing a structure known as a 4E-sensitivity element . In thisArgonautes (AGOs) are the effector proteins functioning in eukaryotic RNA silencing pathways , a sequeTranslational repression guided by sRNAs has been best studied in fly and mammalian cells and is mediated by imperfect base pairing of microRNAs (miRNAs) to target mRNAs . In animin vitro system prepared from plant cultured cells, AtAGO1 has been shown to have the ability to repress translation initiation even when the cleavage of mRNA targets was blocked by introducing central mismatches in the miRNA-target pairing region or mutation of the catalytic core of AtAGO1 .AGO1, AGO2 and AGO10 have been implicated in translation repression  that are subsequently incorporated into antiviral RISCs . AntivirNicotiana benthamiana (Tobacco rattle virus (TRV) in Arabidopsis (N. benthamiana plants infected with Tomato ringspot virus (ToRSV)  early in infection, but reduced RNA2 translation later in infection. Additionally, neither recovery nor the reduction of RNA2 translation were observed in plants silenced for AGO1, suggesting that AGO1 plays a role in the translational repression mechanism targeting ToRSV  . In thisng ToRSV .Potato virus X interacts with AGO1 and mediates its degradation through the proteasome pathway , which disrupt various steps of the silencing pathway . Among tSweet potato mild mottle virus (SPMMV) targets loaded AGO1 and inhibits the si/miRNA-programmed RISC activity. The suppressor/binding activities are localized at the N-terminal half of P1, a region containing three WG/GW motifs (Sweet potato feathery mottle virus (SPFMV) P1, which did not have any silencing suppressor activity, was converted into a VSR by including two additional WG/GW motifs (Turnip crinkle virus (TCV) physically interacts through GW repetitive motifs with unloaded Arabidopsis AGO1  that also encodes the movement protein (MP), both being viral proteins required for systemic infection (The immune receptor NIK1 [nuclear shuttle protein (NSP)-interacting kinase 1] has a remarkable role in the defense response against begomoviruses. It belongs to the receptor-like kinase (RLK) family of plant receptors, and it was first identified as a virulence target of the begomovirus nuclear shuttle protein (NSP)  antiviral machinery and/or salicylic acid (SA) signaling pathway . In the in vitro GST pull-down assays and confirmed in planta through bimolecular fluorescence complementation (BiFC) assays (Cabbage leaf curl virus (CaLCuV) and from tomato-infecting begomoviruses, such as Tomato golden mosaic virus (TGMV), Tomato crinkle leaf yellow virus (TCrLYV) and Tomato yellow spot virus (ToYSV)  belong to the subfamily II of leucine-rich repeat (LRR)-RLKs, designated LRRII-RLK group . NIK1 waNIK function in Arabidopsis is linked to an enhanced susceptibility phenotype to infection by a coat protein-less mutant of CaLCuV , isolated through two-hybrid screening by its capacity to bind to the kinase domain of NIK1 (in vivo translation and decreased loading of host mRNA in actively translating polysomes (PS) fractions. Likewise, induction of T474D expression through a dexamethasone-inducible promoter also impairs global translation, which was correlated with a reduction of both PS and monosome (NPS) fractions, as well as of the RNA content associated with these fractions in the T474D lines. Ectopic expression of T474D controls begomovirus infection, causing symptomless infection, delayed course of infection and reduced accumulation of viral DNA in systemically infected leaves. Additionally, in infected T474D lines, the loading of coat protein viral mRNA in actively translating polysomes is reduced as compared to that of wild type infected lines, suggesting that the translation of viral transcripts is strongly impaired by NIK1-mediated signaling. Thus, begomovirus cannot sustain high levels of viral mRNA translation in the T474D-expressing lines, indicating that suppression of global protein synthesis may effectively protect plant cells against DNA viruses (Tomato severe rugose virus (ToSRV) , which interacts with RPL10 in the nucleus of plant cells  constitutes a promising strategy for plant protection against viruses.


DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.Patients with hypomorphic mutations in  LIG4 that encodes a key component of the ubiquitous non-homologous end-joining (NHEJ) pathway essential for the DNA double strand break (DSB) repair mechanism that is also utilized in the production of T and B lymphocyte receptors.DNA ligase IV deficiency (OMIM 606593) or LIG4 syndrome ORPHA99812), also known as Ligase 4 syndrome, is a rare autosomal recessive disorder characterised by microcephaly, abnormal facial features, sensitivity to ionizing radiation and combined immunodeficiency. Additional features can include developmental delay, bony deformations, skin conditions and susceptibility to malignancy J recombination is recognised by a complex of which the constituent parts are MRE11, RAD50 and Nijmegen breakage syndrome protein 1  . Ku70/808 cells each with their own unique antigen receptor that is able to recognise a single antigen-major histocompatibility complex (MHC) . Inutations . In expeutations .In many patients, there is increased chromosome 7:14 translocation on karyotype analysis. Clonogenic survival assays confirm radiosensitivity, performed by subjecting fibroblasts from the patient to increasing doses of ionising radiation and measuring percentage survival of the cells after a fixed time period . DelayedLIG4 and other DNA repair genes can be performed. To date, no patients with mutations in LIG4 have been described who do not exhibit sensitivity to ionising radiation.Once radiosensitivity is confirmed, specific genetic testing for Until recently LIG4 syndrome was only identified through genetic testing after the diagnosis of immunodeficiency and/or malignancy in microcephalic patients. Murray et al. observed this and took the reverse approach to diagnosis. The authors screened 138 patients with microcephalic primordial dwarfism for DNA Ligase IV mutations. Eleven microcephalic primordial dwarfism patients were identified as having LIG4 syndrome all off whom had cellular radiosensitivity. Nine developed cytopenia due to bone marrow failure requiring transfusion, seven of whom developed cytopenia post-LIG4 screening. Interestingly no patients were diagnosed with CID before screening and only one patient was diagnosed with SCID. Retrospectively a further six patients showed signs of CID, most with increased infection rate, low B-lymphocyte numbers and hypogammaglobulinaemia. Four required HSCT due to significant immunodeficiency. This may be due to the accumulation of DNA-DSB, and therefore progressive apoptosis, in haematopoietic stem cells of LIG4 patients causing progressive immunodeficiency as lymphocytogenesis reduces over time . None ofNBN mutations, the product of which, nibrin, is involved in recognising DNA-DSB [A number of conditions have features that overlap with LIG4 syndrome. Nijmegen breakage syndrome (NBS) patients harbour  DNA-DSB .NHEJ1, who present with similar physical and neurodevelopmental features, CID with T- and B-lymphocytopenia, isotype class switching defects and recurrent infections. Cernunnos-XLF patients, like LIG4, have also co-presented with bony malformations [A few patients with Cernunnos-XLF deficiency are described with mutations in rmations . CernunnThe third enzyme that makes up the NHEJ ligation enzyme complex is XRCC4, which interacts strongly with LIG4 through a tandem BRCA1 carboxyl terminal domain in LIG4 and a coiled-coil region in XRCC4, to form a highly stable complex. A number of patients have recently been reported with mutations in XRCC4. Phenotypically, the clinical presentation is similar to patients with LIG4 syndrome, with characteristic microcephaly and neurodevelopmental delay. Interestingly, given that XRCC4 is required to stabilise LIG4, it is surprising that to date, none of the patients described exhibit clinical immunodeficiency, despite the marked DNA-DSB repair defect. A molecular alteration in repair pattern is described during class switch recombination, but V(D)J recombination appears normal .Fanconi anaemia is characterized by bone marrow failure, often accompanied by other anomalies including skeletal, renal, cardiac and gastrointestinal defects, skin hypo-pigmentation and predisposition to malignancy, particularly leukemia. Most immunological manifestations relate to bone marrow failure, but some patients present in infancy or early childhood with significant or prolonged infections, more consistent with immunodeficiency . MicroceATR-Seckle Syndrome which also presents with microcephaly and “bird-like” facial features occurs due to abnormal function in ATR, a protein that monitors single stranded DNA replication errors at replication forks . HoweverInitial treatment of LIG4 syndrome is supportive, with haematological support of marrow hypoplasia as required, longterm antibiotic, antiviral and antifungal chemoprophylaxis and immunoglobulin substitution. Patients remain at risk of severe infections because of chemo-prophylaxis resistance or compliance issues, and the risk of lymphoid malignancy increases with time. Excessive exposure to ionising radiation should be avoided where possible, and radiographic and computerised tomography should be avoided where possible. Haematopoietic stem cell transplantation is a curative treatment for CID and SCID immunophenotypes, and might reduce the long-term risk of developing lymphoid malignancy due to improved tumour surveillance. Because of the radiosensitivity displayed by LIG4, conditioning regimes should not include irradiation. A low intensity or modified Fanconi anaemia-based conditioning regimen may give the best possible survival, and restoration of normal immunity and marrow hypoplasia, and alkylating agents should be avoided . LongterInformation on haematopoietic stem cell transplantation is available on ten patients , 27, 46 Excellent social care ensures LIG4 syndrome patients maintain a good quality of life. Extra support is required for parents with children with neurodevelopmental delay, children benefit from attending schools with a positive environment for people with learning difficulties .LIG4 syndrome is an extremely rare condition characterised by microcephaly, abnormal “bird-like” facial features, neurodevelopmental delay and immunodeficiencies with radiosensitivity. Specific features that should raise diagnostic suspicion include prenatal microcephaly with growth retardation and developmental delay, marrow hypoplasia, recurrent infection with lymphocytopenia, and hypogammaglobulinaemia, often with a raised IgM. Haematopoietic stem cell transplantation for immunodeficiency can be curative but it is not without its complications, and reduced intensity conditioning regimens should be utilised, with omission of radiotherapy. In both human cases and mouse models, bone marrow failure and immunodeficiencies can be progressive. It may be beneficial to screen patients with clinical characteristics of LIG4 syndrome in order to diagnose and potentially treat immunodeficiency before development of significant sequalae in order that early intervention with antimicrobial prophylaxis, immunoglobulin replacement and possibly HSCT can be considered.


Klebsiella pneumoniae, initial efforts have focused on the detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting Klebsiella surface polysaccharides, the ability to extract information about these antigens is increasingly important. Here we present Kaptive Web, an online tool for the rapid typing of Klebsiella K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyze genome assemblies in a web browser. The results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise. We demonstrate Kaptive Web's utility by analyzing >500 K. pneumoniae genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase-associated clonal group 258 (25 K and 6 O loci). The characterization of a further 309 genomes indicated that such diversity is common among the multidrug-resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable, and accessible typing methods such as Kaptive Web. Kaptive Web is available for use at http://kaptive.holtlab.net/, and the source code is available at https://github.com/kelwyres/Kaptive-Web.As whole-genome sequencing becomes an established component of the microbiologist's toolbox, it is imperative that researchers, clinical microbiologists, and public health professionals have access to genomic analysis tools for the rapid extraction of epidemiologically and clinically relevant information. For the Gram-negative hospital pathogens such as  Whole-genome sequencing (WGS) represents a powerful tool for the characterization and public health surveillance of bacterial pathogens. This technology is now routinely used by a number of public health laboratories –3, and t–4–7–10–Klebsiella pneumoniae is a major cause of health care-associated infections with high rates of multidrug resistance (MDR)  . In parth threat , 15. With threat –19, mono threat ––23, and threat –––26. Sevehreat ––––31. Of n disease –34. Despplex PCR , 36 or Wplex PCR , 38.rfb locus)  , 40–42. ocus) 40–, 43, andocus) 40–, 36. Intocus) 40–, 25, 43,antigens , 44. Thesubtypes , 45. RegKlebsiella polysaccharide capsule is produced through a Wzy-dependent process , so it will report the locus as O1 or O2 on the basis of the presence/absence of these genes as determined by a tBLASTn search with coverage and identity thresholds of 90% and 80%, respectively. If Kaptive finds only one of the two genes, it will report the locus as O1/O2.A complication comes from the fact that O antigens O1 and O2 are encoded by the same two O loci. It is the presence or absence of two other genes elsewhere in the genome, AB819963.1) is available for download via the command-line Kaptive GitHub page (https://github.com/katholt/Kaptive).A Kaptive-compatible O locus reference database comprising the O loci reported in reference http://kaptive.holtlab.net/. It was developed using the web2py framework (https://github.com/kelwyres/Kaptive-Web). Kaptive Web automatically populates the “reference database” selection with the contents of command-line Kaptive's database directory, enabling automatic compatibility with any new locus databases for Klebsiella or other bacteria. Kaptive Web utilizes a 16-core 64-GB RAM server hosted by the Australian NeCTAR cloud. A single genome analysis requires approximately 4 min and 20 s to complete for the K and O locus databases, respectively. The server can run up to 15 analyses simultaneously, enabling large data sets to be processed relatively quickly.Kaptive Web is available for use at ramework . The souK. pneumoniae organisms were obtained as part of the global diversity study  and 12 min (O locus). The results were inspected via the Kaptive Web graphical interface and downloaded in tabular format .Sequence read data for 309 ty study , and 13 ty study  were assr v0.4.1 . Genome Klebsiella genomes (downloaded from GenBank on 12 October 2017) on the basis of ST information generated using Kleborate (https://github.com/katholt/Kleborate). STs 11, 258, 340, 395, 437, 512, 855, and 895 were included in the analyses.The same protocol was used for characterization of 201 publicly available CG258 genome assemblies (see Data Set S3). These genomes were identified among the complete set of Kaptive Web is a browser-based method for running Kaptive and visualizing the results. Users upload one or more assemblies and select their preferred typing database . There iThe locus was found in a single piece  with 100% coverage and 100% nucleotide identity to the reference.The locus was found in a single piece with ≥99% coverage and ≥95% nucleotide identity to the reference, with no truncated/missing genes and no extra genes compared to the reference.The locus was found in a single piece with ≥99% coverage, with ≤3 truncated/missing genes and no extra genes compared to the reference.The locus was found in a single piece or with ≥95% coverage, with ≤3 truncated/missing genes and ≤1 extra gene compared to the reference.The locus was found in a single piece or with ≥90% coverage, with ≤3 truncated/missing genes and ≤2 extra genes compared to the reference.Did not qualify for any of the above.The top two confidence levels, “very high” and “perfect,” require the locus to be found uninterrupted in a single contig with the expected gene content. If the locus has truncated/missing genes or was found in multiple discontiguous pieces, a lower confidence level will result. Insertion sequence (IS) integrations are one possible cause of truncated/missing genes. When an IS interrupts a locus gene, that gene's function is likely lost , and forwzc and wzi K locus genes, for compatibility with earlier schemes that focused on these genes  were caused by a break in the assembly, splitting the locus over two contigs. This may have resulted from poor read coverage, in which case wckR may have been intact and functional in the original isolate. Alternatively, the assembly break may have resulted from an insertion sequence interrupting wckR, in which case, gene function is likely lost; such interruptions have been characterized in a number of Klebsiella K loci , which are distinguishable using genomic data (they differ in terms of gene content) but are serologically cross-reactive. These aspects are incorporated into Kaptive as follows: (i) the relevant O locus variant is reported as v1 or v2, and (ii) an additional search is conducted for wbbY and wbbZ to decide whether the locus should be reported as O1 or O2. If only one of wbbY or wbbZ is found, Kaptive will give a label of O1/O2 .The Kaptive algorithm was originally developed and validated for typing the K locus of eumoniae , but it wbdA and wbdD genes (wbdA (C80R) . The O3dD genes . The O3 A (C80R) . While KKlebsiella global diversity study genomes (wzm and wzt genes (wbbY and wbbZ) and one was where the previous type was O1 and Kaptive assigned O2. The remaining three discrepancies were all between O3 and OL104, which are distinguished by their wbdD genes  (see Data Set S3) and analyzed them using Kaptive Web. “Good” or better K and O locus calls were obtained for 173 (86%) and 186 (93%), respectively (Data Set S3). Importantly, while there were dominant types , there was also much diversity, with 25 K loci and 6 O loci in total (not distinguishing O1 and O2)  .Streptococcus pneumoniae polysaccharide capsule (K. pneumoniae population. Kaptive and Kaptive Web provide simple WGS-based solutions to monitor these trends and ensure that therapies are well targeted and keep up with K. pneumoniae evolution. With its graphical interface and remote computation, Kaptive Web also makes these analyses accessible to the wider public health community.There is now emerging evidence that like CG258, other globally distributed MDR clones also harbor diverse K and O loci , 63. For capsule , 65. The


Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells.Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues.Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis.The online version of this article (doi:10.1186/s12864-016-3060-0) contains supplementary material, which is available to authorized users. Filoviridae and is an envelope, non-segmented, negative-stranded RNA virus with filamentous virion morphology. Infection with Ebola virus causes Ebola Virus Disease, a disease associated with mortality rates between 25 and 90 % [Ebola virus belongs to the family and 90 % . The earand 90 % –3. Durinand 90 % –6.It has been postulated that survival following Ebola virus infection correlates with the ability of the host to mount an early and robust interferon response. In cultured human liver cells, the virus has been shown to block specific aspects of the innate immune response, preventing the expression of interferon-stimulated genes , 8. ExpePrevious studies of primates exposed to wild-type Ebola virus strains have suggested that there is a strong interferon-like transcription signal in circulating immune cells . It has In the present study, we studied the transcriptional profile of circulating immune cells obtained from several different studies. We investigated the most robustly expressed genes that were observed in the peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques following different routes of infection (aerosol and intramuscular), as well as following vaccination of the host. We further investigated whether genes highly-upregulated in PBMCs also showed changes in expression in tissues of infected animals. Our results suggest that interferon-signaling is an early and robust response to Ebola virus infection throughout the body.This RNA sequencing study used cynomolgus macaques divided in two groups: vaccinated and Ebola-naïve . The vac2 and 2.74×102 plaque-forming units (PFU)) 12 PBMC samples were collected at 0, 3, 6 and 8 days post-infection , with an absolute fold change in expression greater or equal to 3, and an average number of reads across all samples greater than 4 counts per million (CPM). When calculating relative changes to the pre-infection samples, infected samples were not subtracted from their individual uninfected controls (since not every infected sample had a pre-infected control), but from an average of all the pre-infection samples. Statistical significance, however, was calculated using the individual pre-infection samples, not their average.A gene was deemed to show statistically significant changes in expression at a specific time after infection if the moderated Agilent two-color human gene expression microarrays were processed using limma . Fold chTo examine the response of the circulating immune system of nonhuman primates infected with Ebola virus, we used sequencing data from PBMCs of cynomolgus macaques infected with Ebola virus via intramuscular injection. These included pre-infection samples and samples taken 4 and 7 days after infection, as described in , 18  CCL7, CXCL11, IL1R1 (cytokine receptor) and IL15RA, and others became upregulated during the late stage of infection (day 7): IL2RA, CSF2RB and others Fig. .The majority of cytokines during Ebola virus infection in vaccinated animals did not reach a 3-fold change in expression at 4 or 7 days post-infection. Three notable exceptions were CCL7, CXCL10 and CXCL11, which peaked (respectively) at 42.8, 35.5 and 6.6 times the level of expression at 7 days post-infection compared to the pre-infection levels. These genes mirrored the expression patterns seen in the Ebola-naïve animals, but at a lower magnitude  [We also analyzed the macaque intramuscular Ebola dataset to identify genes that underwent strong, statistically-significant increases in expression between pre-infection and 4 days post-infection. The thresholds that we used were 8 or higher fold-change with at least 4 read counts per million, and an adjusted or ISGs) , 36  and mouse-adapted EBOV (which is pathogenic in mice) . InteresWe validated these results using expression data from two previous studies that infected rhesus macaques with Ebola virus (intramuscular injection) and treated them with an anticoagulant drug (recombinant human activated protein C (rhAPC), and recombinant nematode anticoagulant protein c2 (rNAPc2) , 38). WeGiven the repeated appearance of a type I interferon-like signature following Ebola virus infection, we were interested in determining whether the appearance of an early innate immune response following a hemorrhagic fever virus infection was a response to the injection of a negative strand RNA virus. To do this, we used the intramuscular Ebola dataset to compare the innate immune response to Ebola virus in non-human primates that had been previously vaccinated against Ebola virus to those that had been immunized against a different hemorrhagic fever virus and were therefore susceptible to Ebola virus (Ebola-naïve group) , 18. FigThe early host transcriptional response that we observed in these studies appeared to be similar to strong innate transcriptional responses observed in other hemorrhagic fevers . To compTo determine if the route of infection could alter the early transcriptional response to Ebola virus infection, we looked at gene expression data from a study that exposed macaques to Ebola virus via aerosol and we compared it to the intramuscular injection data. Figure We next used the list of genes that were strongly upregulated in PBMCs in the macaque intramuscular Ebola dataset  increase between 4 and 7 days post-infection. We also found several cytokines, such as CCL7 and CXCL11 (I-TAC), that showed significant changes in expression but whose protein levels have not been previously observed during Ebola pathogenesis. CXCL10 (IP-10) and CXCL11 were two of the cytokines that were expressed in animals that succumb to infection. Interestingly, we saw a significant increase in expression in vaccinated macaques infected with Ebola virus. This suggests that they are part of a conserved response in both the pathogenic and non-pathogenic response to Ebola virus. Both cytokines are induced by interferon alpha (they are ISGs), share a common receptor (CXCR3), and are thought to be involved in the recruitment of effector T cells and NK cells . They arBy comparing how different routes of infection affect the cellular circulating immune response in Ebola virus-infected primates, we observed that infection via an aerosol and via intramuscular injection resulted in similar patterns of gene expression. Previous studies have reported a delay in the aerosol model of exposure compared to the intramuscular model , but givInfection with Ebola virus leads to early and robust interferon-like responses that take place before the appearance of virus in the blood. This response takes place not only on circulating immune cells, but throughout the majority of infected tissues. Our results extend earlier observations of a strong innate immune response and suggest the involvement of new cytokines in Ebola virus infection. Further analysis of the cells responsible for driving this response, and for producing the different cytokine signals, will be important to understand the ability of the virus to replicate virtually unchecked in many tissues—even when these tissues show a strong interferon-expression signal—, and to identify which cells are undergoing an uncontrolled ISG response.


Macrobrachium rosenbergii causes significant economic losses in shrimp farms and hatcheries and poses a threat to food-security in many developing countries. Outbreaks of Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease (WTD) are associated with up to 100% mortality rates. There are no interventions available to treat or prevent MrNV disease however. Here we show the structure of MrNV virus-like particles (VLPs) produced by recombinant expression of the capsid protein, using cryogenic electron microscopy. Our data show that MrNV VLPs package nucleic acids in a manner reminiscent of other known nodavirus structures. The structure of the capsid however shows striking differences from insect and fish infecting nodaviruses, which have been shown to assemble trimer-clustered T = 3 icosahedral virus particles. MrNV particles have pronounced dimeric blade-shaped spikes extending up to 6 nm from the outer surface of the capsid shell. Our structural analysis supports the assertion that MrNV may belong to a new genus of the Nodaviridae. Moreover, our study provides the first structural view of an important pathogen affecting aquaculture industries across the world.White tail disease in the giant freshwater prawn  Macrobrachium rosenbergii nodavirus (MrNV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. MrNV outbreaks in shrimp farms and hatcheries can result in mortality rates of up to 100%, thus the virus represents a threat to food-security and causes significant economic losses1. WTD disease was first reported in Pointe Noire, Guadeloupe in 1997, and was subsequently detected in China2, India3 Taiwan4, Thailand5, Malaysia6, Australia7 and most recently in Indonesia8.MrNV belongs to the family Nodaviridae, which is divided into Alphanodavirus and Betanodavirus genera. The former includes insect-infecting nodaviruses such as Nodamura virus (NoV), Boolaara virus (BoV), Flock House virus (FHV), black beetle virus (BBV) and Pariacoto virus (PaV). The latter comprises fish-infecting nodaviruses such as Malabaricus grouper nervous necrosis virus (MGNNV)  at 150,000 × g for 4.5 hours at 4 °C with a SW 40 Ti rotor . This was followed by fractionation into 1 ml fractions. The fractions which contained the MrNV capsid protein, as analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), were dialysed in sodium phosphate buffer as above. The purified protein was concentrated by centrifugation using a centrifugal concentrator with molecular cut-off 50 kDa  at 4000 × g at 4 °C. The final protein concentration was determined by the Bradford assay.MrNV capsid protein at approximately 1 mg/ml was prepared for cryogenic transmission electron microscopy using an FEI Vitrobot Mk IV. Four μl of VLP preparation was applied to freshly glow-discharged quantifoil holey carbon support films , blotted for 4 seconds and plunged into liquid ethane36. Vitrified samples were viewed at low-temperature (around 98 K) and under low electron dose conditions using a JEOL 2200 FS cryo-microscope operated at 200 kV. Samples were held in a Gatan 626 cryo-stage. Micrographs were recorded at 50,000 × magnification on a Direct-Electron DE20 DDD camera giving a sample rate of 1.09 Å/pixel. Three-second exposures, at an electron dose rate of ~20 e/Å2/s, were captured in movie mode running at 20 frames per second.Purified 37. Initially movies were corrected for specimen movement using MotionCorr38 and defocus was estimated using GCTF39. An initial set of ~1000 particles was manually picked and processed to calculate a 2D average suitable for automated particle picking. Following automated particle picking 2D classification was used to select intact particles for 3D reconstruction. Representative 2D class-averages are shown in Supplemental Fig. 40. In line with recommended procedures the starting model was low-pass filtered to 60 Å to limit the effects of model-bias. A subset of particles was selected from the 3D class that yielded the highest resolution and further refined. Following refinement, per-particle movie correction was performed using only frames 2–20, to limit the impact of radiation damage. Finally, a post-processing b-factor of −890 Å2 was estimated and applied to the reconstruction using Relion41. To satisfy ourselves that our model was not affected by model-bias, we also performed a 3D autorefine analysis of our final dataset using a Gaussian sphere as a starting model, yielding essentially identical results (Supplemental Fig. Micrograph movies containing 60 frames were processed to calculate a 3D reconstruction using Relion-2.042. The map has been deposited in the electron microscopy data bank, with accession number EMD-3655.The calculated 3D reconstruction was visualized in UCSF chimera and IMODSupplemental figures 1 to 4


Evidence from population-based studies on the economic burden imposed by chronic non-communicable diseases (CNCDs) is still sparse in Sub-Saharan Africa. Our study aimed to fill this existing gap in knowledge by estimating both the household direct, indirect, and total costs incurred due to CNCDs and the economic burden households bear as a result of these costs in Malawi.The study used data from the first round of a longitudinal household health survey conducted in 2012 in three rural districts in Malawi. A cost-of-illness method was applied to estimate the economic burden of CNCDs. Indicators of catastrophic spending and impoverishment were used to estimate the economic burden imposed by CNCDs on households.A total 475 out of 5643 interviewed individuals reported suffering from CNCDs. Mean total costs of all reported CNCDs were 1,040.82 MWK, of which 56.8 % was contributed by direct costs. Individuals affected by chronic cardiovascular conditions and chronic neuropsychiatric conditions bore the highest levels of direct, indirect, and total costs. Using a threshold of 10 % of household non-food expenditure, 21.3 % of all households with at least one household member reporting a CNCD and seeking care for such a condition incurred catastrophic spending due to CNCDs. The poorest households were more likely to incur catastrophic spending due to CNCDs. An additional 1.7 % of households reporting a CNCD fell under the international poverty line once considering direct costs due to CNCDs.Our study showed that the economic burden of CNCDs is high, causes catastrophic spending, and aggravates poverty in rural Malawi, a country where in principle basic care for CNCDs should be offered free of charge at point of use through the provision of an Essential Health Package (EHP). Our findings further indicated that particularly high direct, indirect, and total costs were linked to specific diagnoses, although costs were high even for conditions targeted by the EHP. Our findings point at clear gaps in coverage in the current Malawian health system and call for further investments to ensure adequate affordable care for people suffering from CNCDs.The online version of this article (doi:10.1186/s12913-016-1716-8) contains supplementary material, which is available to authorized users. Chronic non-communicable diseases (CNCDs), defined as non-communicable conditions that affect people in the long term , accountThe World Health Organization (WHO) predicts that by 2030 the largest proportional rise in CNCD deaths globally will occur in Sub-Saharan Africa (SSA), where the disease burden of CNCDs is already very large , the incStill, compared with communicable diseases, researchers have paid limited attention to the economic burden imposed by CNCDs in SSA . The mosThis population-based study was designed to fill this existing gap in knowledge by describing direct, indirect, and total costs for the most frequently reported CNCDs and the related economic impact of these conditions on affected households in rural Malawi.In recent years, CNCDs have been causing an increasing number of deaths and disabilities in Malawi . In 2008Health service provision in Malawi relies on a mixture of public and private providers. Government funded health provision consists of a combination of tax-based and donor financing to ensure that services included in the Essential Health Package (EHP) are provided free of charge at point of use, at either public or contracted private not-for-profit facilities. The EHP is meant as a measure to advance progress towards universal coverage and as such includes most priority health interventions targeting the major causes of disease burden in the country . In 2010n = 691) and households where no person was a SACCO member (n = 525). The total sample included 5643 individuals distributed in 1199 households. The panel survey questionnaire collected information on both household socio-demographic and economic characteristics, on individual illness profiles, and on the specific health seeking behavior (including costs) of people suffering from acute and chronic conditions . In addition, there are 6 private for-profit facilities. Details of the sampling procedures for the panel household survey have been described elsewhere . In briePeople suffering from at least one CNCD were identified in two steps. First, we asked respondents whether they suffered from any chronic disease, which we defined as any illness or complaint that persisted in an individual longer than three months, or any recurrent non-communicable illness episodes that had initially started in the past and continued to affect a respondent’s health by the time of interview . In a seOnce identified as suffering from a chronic condition, individuals were asked a series of additional questions pertaining to their health seeking behavior and related direct and indirect costs. In consonance with other researches –29, in oWe defined the primary outcome variables for this study as the direct and indirect costs as well as the sum of these two costs, i.e. total costs, incurred by an individual due to CNCDs. All cost values are expressed in the local currency, Malawian Kwacha (MWK) (1USD ~ 280MWK at the time of survey). In addition, based on the analysis of direct costs only, we looked at catastrophic spending and impoverishment due to CNCDs.We defined direct costs as the out-of-pocket expenditure arising in the process of seeking care, including direct medical and direct non-medical costs . In thisIn line with prior research  using thThe total cost of illness due to CNCDs was estimated by simply adding direct and indirect costs for all individuals who reported at least one CNCD. When calculating total costs, direct costs for those who did not seek any care and indirect costs for those who were younger than 10 years or older than 64 years were counted as zero .We defined catastrophic spending due to CNCDs as the share of household out-of-pocket health expenditure caused by CNCDs in relation to household non-subsistence expenditure  once exceeding a given threshold level . In pracWe defined impoverishment due to CNCDs as the difference in poverty rate before paying for CNCD care (i.e. pre-payment poverty rate) and after making such payments (i.e. post-payment poverty rate), i.e. the percentage of non-poor households falling into poverty due to direct costs associated with CNCDs , 39. DueDue to the uneven distribution of direct, indirect, and total costs, we used mean, median, and inter-quartile range to describe the central tendency of each of these cost estimates. The incidence of catastrophic spending due to CNCDs was computed as the proportion of households incurring catastrophic spending in relation to the total number of households reporting CNCDs , 42. We n = 126, accounting for 24.5 % of all reported conditions), chronic respiratory conditions , chronic cardiovascular conditions , chronic sense organ conditions , chronic neuropsychiatric conditions , and chronic digestive conditions . Chronic skin or oral conditions, malignant neoplastic conditions, chronic genitourinary conditions, and chronic endocrine conditions were excluded from further analysis due to the small number of respondents in these categories. Among the six most frequently reported conditions, only chronic cardiovascular conditions were covered by the EHP at the time of the study. Out of these 475 respondents reporting at least one CNCD, 298 (62.7 %), distributed in 244 households, sought some form of either formal  or informal care  during the four weeks preceding the interview, while 320 (67.4 %) were of 10 to 64 years of age.Out of 5643 individuals included in the survey, 475 (8.4 %) reported a total of 515 chronic non-communicable conditions . Out of the ten disease categories used in our study to group chronic complaints, the six most frequently reported were: chronic pains  and 1,617.83 MWK  respectively. Mean monthly direct costs were lowest for individuals with chronic sense organ conditions and chronic digestive conditions, at 410.31 MWK  and 416.38 MWK , respectively , while those who reported chronic sense organ conditions and chronic respiratory conditions bore the lowest mean monthly indirect costs   and lowest for individuals reporting chronic sense organ conditions and chronic respiratory conditions (465.35 MWK and 623.60 MWK respectively) (Table Table Table To our knowledge, this is one of the very first studies exploring the economic burden imposed on SSA households by a broad range of CNCDs, with information derived from population-based rather than a facility-based sample. Our findings indicate that CNCDs carry a relatively high economic burden for the local population, which causes financial stress and aggregates poverty in rural Malawi. In addition, our study identified remarkable differences in economic burden depending on the specific condition affecting an individual. Considering the scarcity of cost-of-illness studies on CNCDs in SSA, we discuss our findings in relation to literature emerging from other LMICs as well.Our study found that direct costs accounted for the largest portion of the total cost of illness across a broad range of CNCDs, whereas direct medical costs comprised the largest portion of direct costs to the individual as a result of these conditions. These findings are consistent with cost-of-illness studies on cervical cancer  and on hAlso in line with prior evidence , 16, 46,Among the most frequently reported CNCDs, we showed that individuals affected by chronic cardiovascular conditions and chronic neuropsychiatric conditions bore the highest levels of direct, indirect, and total costs. This finding is in line with the report on the global economic burden of non-communicable diseases recently released by Harvard University . Also, cThe fact that chronic cardiovascular conditions resulted in the highest level of direct costs appears even more striking, given that these conditions are covered by the EHP and related services should be provided free of charge at point of use. Again in line with prior qualitative evidence , our finOur findings also revealed that a considerable proportion of households incurred catastrophic spending due to CNCDs. Poorest households faced the highest risk of catastrophic spending due to CNCDs. These findings, which rely on direct costs for CNCDs aggregated at the household level, support findings from our previous work, which pointed at the regressivity of individual out-of-pocket expenditure on CNCDs . These fIn addition, our study found that an additional 1.7 % of households with at least one household member reporting a CNCD fell under the international poverty line once expenditures due to CNCDs were considered. The fact that the economic burden of CNCDs aggravates poverty is in line with previous studies focusing specifically on the economic impact of CNCDs on affected households , 18 and At last, we need to consider a few limitations of our study. First, as mentioned in the methods section, we could not estimate caretakers’ productivity losses, due to absence of relevant information from the survey. In addition, our estimation of direct costs is likely to be lower than their true value, since we did not have data on other direct non-medical costs, such as food and accommodation expenses incurred by the sick individuals and their caretakers. Thus, both direct and indirect costs resulting in the overall economic burden are likely to be higher than what estimated in our study. Second, the individual’s earnings are not easily traceable in subsistence societies where people produce most of what they will consume . This reOur study showed that in rural Malawi, the economic burden of CNCDs remains high, leading households to face catastrophic spending and pushing households further into poverty. This finding is worrisome given that in Malawi, health care services are set to be provided free of charge at point of use for a broad range of conditions, including several chronic non-communicable ones. Our findings further indicated that particularly high direct, indirect, and total costs were linked to specific diagnoses, but that in general costs were high even for conditions in principle covered by the EHP. These findings point at clear gaps in financial protection and call for further investments, not only increasing effective coverage of chronic conditions via the EHP, but also expanding formal social health protection mechanisms, such as offering monetary compensation for work lost in case of illness. Further studies are needed to replicate and thus validate our research findings in other SSA settings.