Woad root has been used for the prevention of influenza for hundreds of years in many Asian countries. In this study, the antiviral modes of clemastanin B (CB), epigoitrin, phenylpropanoid portion (PEP), and the mixture of phenylpropanoids, alkaloids, and organic acid portions (PEP + ALK + OA) from wood root extract against influenza virus A FM1 were investigated. The results revealed that CB, epigoitrin, PEP, and PEP + ALK + OA exert their anti-influenza activity via inhibiting the virus multiplication, prophylaxis, and blocking the virus attachment. The primary mode of action of PEP and PEP + ALK + OA is the inhibition of virus replication. The inhibitory effect on virus attachment and multiplication is the main modes for epigoitrin. All the compounds or chemical portions from woad root extract tested in this study do not have direct virucidal activity. Our results provided the comprehensive analysis of the antiviral mechanism of wood root extract.  Influenza or flu is one of the most significant acute respiratory diseases caused by the infection of influenza virus. Seasonal influenza affects millions of people in the world every year, leading to a serious threat to public health especially to children and the elderly. In addition, influenza virus has the potency to cause a severe pandemic and economic loss . The outCurrently, the synthetic antiviral drugs or vaccines have limited use in developing countries due to the emergence of resistant strains, the high cost, and the harmful side effects , 3. Howe Isatis indigotica Fort. Woad root was first documented as the herbal drug in The Divine Husbandman's Herbal Foundation Canon, a famous ancient medical book in the Han Dynasty of China (200 AD). It has been used in the treatment of cold, sore throat, and headache for hundreds of years in China , 1413, 1Mouse lung-adapted variant of influenza virus A FM1 strain was obtained from the Department of Microbiology and Immunology at Shandong University. The virus was propagated twice in the allantoic cavity of 9- to 10-day-old embryonated hen's eggs at 35°C for 48 h to enhance the virulence. The allantoic fluid was harvested for the measurement of its hemagglutinating activity. Once the hemagglutination titer reached 1 : 640, the virus was aliquoted and stored at −80°C until use. The Madin-Darby canine kidney (MDCK) cells and human cervical cancer (HeLa) cells were obtained from Institute of Cell Biology, Chinese Academy of Sciences. The positive control ribavirin (Batch number: 101018) was purchased from Baili Pharmaceutical Co. Ltd. in Sichuan province, China.μm membrane. For those compounds which cannot be dissolved in the medium, they were first dissolved in DMSO and then diluted with the fresh medium. The final DMSO concentration in the medium was less than 1%. The concentration of CB and epigoitrin was 50 μg/mL and the initial concentration of PEP and PEP + ALK + OA was 100 μg/mL.The woad root was collected from Anhui province, China. Herb identification was confirmed through morphological and microscopic analysis according to the Chinese Pharmacopeia . The extThe anti-influenza action of CB, epigoitrin, and chemical portions from woad root extract was investigated in four different modes: therapeutic action, prophylaxis, direct virus inactivation, and inhibition of virus attachment.The cells were preinfected with the virus before the pure compounds or chemical portions of plant extract were added. The therapeutic action of the drugs was evaluated by both cytopathic effects (CPE) reduction assay and cell MTT assay.50 (50% Tissue Culture Infective Dose) of virus for 2 h. The unabsorbed virus was then washed off using PBS. Quadruplicate cell monolayers were subsequently overlaid with 0.1 mL medium containing different nontoxic twofold serial dilutions of pure compounds, chemical portions of woad root extract. Cells with virus infection without drug treatment and the cells without virus and drugs were used as controls. The plates were incubated at 37°C under 5% CO2 for 72 h. The virus-induced CPE was observed under a light microscope in comparison with the parallel virus control and cell control.The CPE reduction assay was conducted according to the previous report with slight modifications . BrieflyμL of MTT was added to each well and incubated at 37°C for 4 h. Subsequently, DMSO was added and the absorbance was measured at 570 nm. The cells protection rate (%) was calculated by the following formula:The MTT reduction assay was performed according to the standard protocol . In shor100%.See .50 of influenza virus A FM1 at 37°C for 2 h to allow virus absorption. Subsequently, the unabsorbed virus was washed off using PBS and the equal amount of maintenance medium was added into each well. The plates were incubated at 37°C under 5% CO2 for 72 h. The virus-induced CPE was observed under light microscope.To evaluate the effects of pure compounds and chemical portions from woad extract on prophylaxis of cell infection, the MDCK monolayer cells in 96-well plates were overlaid with different nontoxic twofold serial dilutions of pure compounds, chemical portions of woad root extract. Four replicates were set up for each treatment and control. After 4 h, the test substances were removed from the wells and the monolayer cells were then infected with 100 TCID50 of the virus was treated with equal volumes of twofold diluted pure compounds or extract portions for 2 h at 37°C. The samples were then tenfold serially diluted. When the confluent monolayer of MDCK cells was formed, the surviving virus in the mixtures was determined in CPE assay and titers  were calculated according to the Reed-Muench method.The direct virucidal activity of the pure compounds and chemical portions from woad extract was tested according to the methods described by Carlucci et al. . One hun50 of the virus were simultaneously added to MDCK cells [2 for 72 h. The virus-induced CPE was observed under light microscope.The monolayer of MDCK cells was cultured in 96-well plates. One hundred microliters of different nontoxic twofold serial dilutions of pure compounds, chemical portions of woad root extract, and the equal volume of 100 TCIDCK cells . After it-test was performed to compare between the control and treatments. A value of p < 0.05 was considered as significant difference (∗) and p < 0.01 was considered very significant (∗∗).All the experiments were repeated three times, each with quintuplicate determinations. The data were expressed as mean ± SD. Student's http://dx.doi.org/10.1155/2016/2537294). However, MDCK cells grow well in the drug treatment groups . The therapeutic action was evaluated by both CPE assay and MTT assay. Clear cytopathic effects were observed in MDCK cells infected with FM1 after 72 hours such as increased gaps between cells, rupture of the cell nucleus, and the partial or complete collapse of cells (p < 0.01) (p < 0.05). This indicated that compounds and extract portions from woad root have better therapeutic action against influenza A virus FM1 than the current commercial synthetic antiviral drug ribavirin. Additionally, the protective effect of CB, epigoitrin, PEP, and PEP + ALK + OA portions was not dose-dependent. The highest protection rate was observed in 1 : 4 dilution of CB, epigoitrin, or the mixture of PEP + ALK + OA portions, while PEP diluted 1 : 8 resulted in the highest cell viability  . The moliability . In comp50. The CPE formation was observed and the cell viability was measured by MTT assay. CPE assay showed that there was no obvious CPE formation in MDCK cells pretreated with CB, epigoitrin, PEP, and PEP + ALK + OA portions in all the dilutions (Table S2).In order to evaluate the effects of pure compounds and chemical portions from woad root extract on prophylaxis of influenza A virus, the MDCK cells were treated with serial dilution of CB, epigoitrin, PEP, and PEP + ALK + OA portions, respectively. After 4 h, the drugs were removed from the wells and the cells were infected with 100 TCIDp < 0.01). Moreover, compared with ribavirin, natural compounds or extracts from the wood root have higher prophylactic activity against influenza virus A FM1 (p < 0.01). The cell viability was dose-dependently increased by PEP and PEP + ALK + OA portions . The inhibition of virus adsorption to the host cells by CB, epigoitrin, and PEP was in a dose-dependent manner. The maximum inhibitory effect of CB, epigoitrin, and PEP was observed with a 1 : 2 dilution, which resulted in 3.53, 3.99, and 4.43 times' increase in cell viability, respectively. In contrast, the highest cell protection rate in PEP + ALK + OA and positive control ribavirin groups was found in a 1 : 4 dilution. Additionally, it was shown that CB, epigoitrin, PEP, and PEP + ALK + OA are more effective than ribavirin on the inhibition of influenza A FM1 virus attachment  in the supplementary materials describe the formation of CPE (Cytopathic effect) in MDCK cells after the treatment of woad root extract. The cells were infected with pre-infected with Influenza Virus A FM1. Cells were then treated with the chemical portions and pure compounds from woad root extract in three different modes including therapeutic action, prophylaxis and direct virus inactivation.


To the Editor:Cryptococcus gattii is a fungal pathogen that is emerging in the Pacific Northwest of North America. In Nova Scotia, Canada, previously not recognized as a C. gattii–endemic area, a variant strain similar to VGIIb caused cryptococcosis with nasopulmonary, lymph node and central nervous system involvement in a free-ranging, yearling white-tailed deer (Odocoileus virginianus). The deer was found in the village of Greenwood  on July 14, 2014. The deer exhibited behavioral and neurologic abnormalities, including loss of fear of humans, ataxia, circling, high-stepping gait, torticollis, and a fixed stare. Additional clinical signs were ptyalism with frothing from the mouth and dyspnea with gurgling respiration. The animal was euthanized, and the head, lungs, heart, gastrointestinal tract, liver, and kidneys were submitted for pathologic examination.Gross examination revealed multifocal, soft, round, expansile, pale tan masses of variable sizes that had replaced or effaced the normal architecture of the tracheobronchial lymph nodes and pulmonary parenchyma. The center of the largest lymph node mass was necrotic and filled with viscous yellow material. Similar yellow gelatinous material obliterated the right ethmoturbinates rostral to the cribriform plate. In the brain, cerebellar coning was prominent. Several small, pitted lesions with dark rims were noted in the neuropil of the thalami, superior colliculi, and hippocampus. Gross lesions were absent in the liver, kidney, and gastrointestinal tract .Cryptococcus  yielded 8 diagnostic signals consistent with C. gattii VGIIb and VGIIc; therefore, the isolate was further classified by multilocus sequence typing based on 7 genetic loci, following the International Society for Human and Animal Mycology consensus multilocus sequence typing scheme for the C. neoformans/C. gattii species complex . However, because of a slight difference in the SOD1 allele , this strain is considered to be a unique variant strain, most similar to that of the VGIIb genotype. Whole-genotyping studies have provided evidence of multiple distinct introductions of the VGIIb genotype to North America . In eastern North America, the C. gattii VGIIb genotype is reported to have caused disseminated cryptococcosis in a human in Florida, USA (C. gattii is potentially pervasive in the environment, the Nova Scotia Department of Health has alerted provincial infectious disease specialists and the provincial public health laboratory to ensure availability of the diagnostic capacity to test for the fungus. The white-tailed deer represents a new host species for C. gattii VGIIb genotype causes substantial, life-threatening disease in otherwise healthy hosts (The Cryptococcus gattii VGIIb-like variant, Nova Scotia, Canada.Gross pathologic findings in white-tailed deer infected with


Ligula intestinalis (Diphyllobothriidea) is one of the most fascinating cestode parasites because it may cause parasitic castration of its second intermediate host, teleost freshwater fishes, due to inhibition of production of fish gonadotropic hormones. Large-sized (length up to 1 m) larvae called plerocercoids develop several months in the body cavity of freshwater fish and affect host behavior to facilitate transmission to the final host, a fish-eating bird. Vitellogenesis, i.e. formation of vitellocytes, is a key process in formation and nutrition of female gametes, oocytes in many flatworms, mainly parasitic Neodermata. The present study provides the first ultrastructural evidence in flatworms (Platyhelminthes) of the process that is interpreted as cytoplasmic-like cell death, i.e. a special case of programmed cell death (paraptosis) in vitellocytes of L. intestinalis.The tapeworm L. intestinalis share numerous features in common with the members of other earliest evolved eucestodes.As molecular markers for paraptosis are not yet available, its identification was based on morphological criteria. Electron microscopy analyses revealed evident structural changes in vitellocytes associated with progressive cytoplasmatic vacuolation, swelling of the granular endoplasmic reticulum and mitochondria. In addition, the present study has shown that vitellocytes of The present study indicates that paraptotic-like cell death may occur in parasitic flatworms (Neodermata). The presence of GER-bodies in mature vitellocytes indicates close relationship between the Diphyllobothriidea, Caryophyllidea and Spathebothriidea, which are considered as the earliest evolved groups of the Eucestoda. Beyond the general similarities, however, a number of differences exist between the morphology, chemical composition and amount of these inclusions which could be due to the variations in their embryonic development, life cycle strategies and definitive host groups. Ligula intestinalis  (Cestoda: Diphyllobothriidea) is a worldwide distributed tapeworm of veterinary importance that affects cyprinid fishes impeding their reproduction by parasitic castration [L. intestinalis has not yet been discovered, this tapeworm has also attracted attention because of detection of several genetic lineages that indicate the existence of cryptic species [Plerocercoids may influence behaviour of their fish hosts and significantly reduce their fecundity or even lead to castration –3. This  species , 5. Ther species ), but no species , 8, whic species .Recently, a series of ultrastructural studies on vitellogenesis in cestodes has been published, including two papers focused on species of the order Diphyllobothriidea , 11. TheL. intestinalis  were obtained from the intestine of the freshly killed great crested grebe Podiceps cristatus (Linnaeus) (Aves: Podicipediformes) collected in Záhlinice, North Moravia, Czech Republic on 20 April 2007 . Live worms were rinsed in 0.9 % NaCl solution and processed for transmission electron microscopy (TEM). Mature and gravid proglottids were separated and fixed with cold 2.5 % glutaraldehyde in cacodylate buffer. After 10 days in the fixative, samples were rinsed overnight in 0.1 M sodium cacodylate buffer at pH 7.4, postfixed in cold (4 C) 1 % OsO4 in the same buffer for 1 h, dehydrated in a graded series of ethanol and propylen oxid, embedded in Araldite and Epon and polymerized at 62 °C for 48 h. Ultrathin sections (60–90 nm in thickness) through selected regions were cut with diamond knife on a Leica Ultracut UCT ultramicrotome and placed on copper grids. Post-processing included staining with uranyl acetate and lead citrate according to Reynolds [Adults of Reynolds . The obsL. intestinalis show high nucleus/cytoplasmic ratio , Golgi complex, spherical and oblong mitochondria. Ovoid nucleus contains a nucleolus and electron-dense patches of heterochromatin Fig. . AccumulA few lipid droplets are formed in the cell cytoplasm, which is already filled with shell globule clusters during the advanced stage of vitellocyte maturation Fig. . They arThe cytoplasm of mature vitellocytes contains shell globule clusters, lipid droplets, glycogen granules scattered between the lipid droplets and GER-bodies Figs. , 4e. StrEvident decrease in the number of lipid droplets and changes in their electron-density may be observed at this stage of vitellocyte differentiation , Cephalochlamys namaquensis , Duthiersia expansa Perrier, 1873 and Schistocephalus solidus  (see [The process of vitellogenesis in 76) see , 11). Th. ThL. inHowever, conspicuous differences exist in the form and number of vitelline inclusions, which may vary between species of the same dipyllobothriidean families . This isL. intestinalis showed no peculiarities, wherein immature and maturing vitellocytes have several features in common with that of other diphyllobothriidean cestodes, i.e. the cytoplasm matrix is abundant of cell organelles participating in synthesis and secretion of shell globules and the formation of shell globule clusters which take part in the egg-shell formation. However, there is high intraspecific variation in the size and number of shell globules and morphology of shell globule clusters.In general, early stages of vitellocyte development in L. intestinalis show differences when compared with those of other diphyllobothriidean species. The main differences lie in the type and amount of lipid droplets and the presence or absence of atypical vitelline inclusions.In contrast, advanced and mature vitelline cells of A recent study on vitellogenesis in the Diphyllobothriidea by Yoneva et al.  has showL. intestinalis and S. solidus during their maturation. Młocicki et al. [Wenyonia virilis Woodland, 1923 and Khawia sinensis Hsü, 1935, parasites of freshwater teleosts, could represent the waste metabolic products of the early embryos. In contrast, reduced quantity of lipids in the cyclophyllidean cestode Mosgovoyia ctenoides   indicates that they may serve as energy reserves [L. intestinalis represent essential nutritive reserve for the embryo or waste products of metabolism.Smyth and McManus  proposedi et al.  and Bruňi et al.  assumed Glycogen is one of the major energy supplies in vitellocytes of cestodes. It occurs both as aggregates or rosettes (α-particles) for long-term storage and/or as discrete granules (β-particles) for rapid utilization. The type and amounts of glycogen reserves accumulated in the mature vitellocytes is highly variable in different group of cestodes, but β-particles apparently represent the most common form observed in cestode vitellocytes. The highest accumulation of glycogen was observed in the members of the order Caryophyllidea where high amount of glycogen granules was observed not only in their cytoplasm but also in the nucleus –21. The D. latum and S. solidus, which use homeotherm vertebrates as their definitive hosts, contain a significantly higher accumulation of glycogen granules compared to those of D. expansa and C. namaquensis, whose adults mature in reptiles and amphibians, respectively, i.e. poikilotherm definitive hosts [Ligula intestinalis matures in homeotherm definitive host (a fish-eating bird), but its vitellocytes contain only a small amount of glycogen granules randomly distributed throughout the vitelline cytoplasm. This indicates that the volume of glycogen in vitellocytes may not be always directly correlated with the type of definitive host.Our observations also reveal that the amount of glycogen varies between diphyllobothriidean species  seem to be present in most species of the earliest evolved eucestode orders, i.e. Caryophyllidea, Spathebothriidea and Diphyllobothriidea Our data on four species of diphyllobothriideans clearly show that the formation of atypical lamellar inclusions in the cytoplasm of mature vitellocytes coincides with the breakdown of the granular endoplasmic reticulum  [The same structures were also observed in the egg-enclosing vitellocytes of the caryophyllidean cestodes  Pallas, 81 [17, 2s, 1781) , 25. Thes, 1781) . FurtherL. intestinalis during the present study. Vitelline cells of L. intestinalis undergo extensive cytoplasmic vacuolation involving different structures and organelles. The appearance of vacuoles, GER and mitochondrial swelling in almost all cytoplasmic area resembles some of the characteristic morphological features of cytoplasmic cell death known as paraptosis, which is one of several types of programmed cell death (PCD) [Various morphological changes were observed during differentiation of vitellocytes of th (PCD) . PCD is th (PCD)  to descrth (PCD) . Howeverth (PCD) . It is ath (PCD) . Thus neL. intestinalis during their development undergo changes, in order to achieve removal of non-functional cells, which might be considered as cytoplasmic-like cell death or paraptosis. This assumption is supported by the formation of characteristic paraptotic features, such as: cytoplasmic vacuoles (predominantly from the endoplasmic reticulum), GER and mitochondrial swelling, as well as the absence of characteristics typical for apoptosis, i.e., nuclear fragmentation, cellular blebbing and apoptic body formation. This is in agreement with observations made by Hoa et al. [In the present study, based on morphological evidence, we hypothesize that vitellocytes of a et al.  who demoL. intestinalis vitellocytes.According to literature paraptosis is characterized by the presence of cytoplasmic vacuolation without nuclear fragmentation that begins with swelling of the endoplasmic reticulum and/or mitochondria , 28. ApoUntil now, there are no data that would indicate the existence of cytoplasmic cell-like death in flatworms (Platyhelminthes). Therefore, this is the first observation that provides ultrastructural evidence of cell changes that are interpreted as results of paraptotic-like cell death.L. intestinalis) may represent an interesting organism for studies focused on the understanding the process of cytoplasmic-like cell death. As this characteristic type of PCD may represent ancestral form of programmed cell death [Our results may suggest that some of the cestode species (as ll death , its preOur results show that despite finding close morphological similarity among representative species of all diphyllobothriideans related with different host groups, no definite conclusion could be made regarding the possible phylogenetic implications due to the limited available data. However, the presence of atypical vitelline inclusions in mature vitelline cells, i.e. GER-bodies indicates close relationship between the Diphyllobothriidea, Caryophyllidea and Spathebothriidea, which are considered as the earliest evolved groups of the Eucestoda.


Pestivirus genus of the family Flaviviridae, was determined here. The virus, originally isolated from a pronghorn antelope, has a genome of 12,273 nucleotides, with a single open reading frame of 11,694 bases encoding 3,897 amino acids.The complete genome sequence of pronghorn virus, a member of the  Pestivirus genus of the family Flaviviridae is composed of a group of agriculturally important viruses. The recognized species of the genus include bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, classical swine fever virus, and border disease virus (pro), the hallmark of the pestiviruses. The pronghorn isolate belongs to the noncytopathic biotype and grows in both ovine- and bovine-derived cell lines. Pronghorn virus was considered the most divergent pestivirus until the isolation and sequencing of Bungowannah virus  individual sequences, with an average depth of coverage of 712.6×. Illumina sequencing provided 4,340,978 reads with 700,290 pestivirus sequence reads. The pestiviral sequences were assembled in one single contig, with a median coverage of 4,290.5×. Following assembly of the complete genomic RNA using data from both platforms, the genome length was determined to be 12,273 bases, with a single ORF of 11,694 bases . The 5′ and 3′ untranslated regions (UTR) comprise 369 and 210 nucleotides, respectively. These are similar in length to those reported for other pestiviruses. There are seven nucleotide differences between the sequences determined by the two methods  that most likely resulted from subculturing of the virus. In addition, two nucleotide residues within the 3′ UTR (A12257 and T12260) were absent in the genomic sequence determined by Illumina sequencing, which most likely resulted from subculturing of the virus. In the earlier report describing pronghorn virus, the nucleotide sequences of NAY781152.The complete genomic sequence of pronghorn virus has been deposited in GenBank under the accession no.


HCO3− and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3- remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JThe online version of this article (doi:10.1007/s00424-014-1543-x) contains supplementary material, which is available to authorized users. In  kinases . Loriol xchanger , 47 withpendence , but con- ions pass and are co-secreted with the protons during gastric acid secretion, the fact that newborn mice had normal acid secretory rates argues for caution with this conclusion. In the airways, Slc26a9 deletion resulted in loss of the short-circuit current (Isc) increase which was seen in the bronchi excised from mice that have been subjected to IL-13-mediated airway inflammation [sc response to cAMP- and Ca2+-dependent stimulation was not affected [We previously reported an expression of Slc26a9 in gastric parietal cells, and a progressive postnatal loss of parietal cells and acid secretory capacity, resulting in achlorhydria by 6 weeks of age in Slc26a9-deficient mice . While tslc26a9 gene have been found to be associated with an increased risk for meconium ileus in infants with cystic fibrosis [Recently, polymorphisms in the fibrosis . We therSlc26a9-deleted mouse strain, whose establishment and characteristics have been described elsewhere [Slc26a3-deleted mice, congenic on the C57/B6 background, were used, which had been bred as recently described [The lsewhere , was conescribed . The Slcescribed . We alsoescribed  and CFTRlater® (Life Technologies) and homogenized. The protocols were approved by the Hannover Medical School Ethics Committee.Informed consent was obtained prior to endoscopy. In addition to the biopsies for routine histology, an additional biopsy was taken from the fundus, antrum, and duodenum of patients that underwent upper GI endoscopy or from different parts of the colon  as well as the terminal ileum of patients that underwent lower GI endoscopy. We selected patients with a healthy upper or lower digestive tract, respectively (neither macroscopic nor microscopic changes). The biopsy was rinsed in phosphate-buffered saline, immediately transferred to RNAlater, as described previously [Mucosa was scraped from the different intestinal segments from WT mice and immediately homogenized in RNAeviously . RNA wasFor microdissection of crypt and villous duodenocytes from Slc26a9 WT mice, tissue collection and fixation were performed as described by Goldworthy et al. . The lasSurvival statistics were performed for mice after the weaning period, by following spontaneous death over a period of up to 1 year. Within a short time span after death, macropathological examination was performed to check for the occurrence of intestinal obstruction, which was the cause of death in CFTR-deficient mice after weaning. The incidence rate of this complication can be lowered to but not fully prevented by dietary measures. Occasional long-term survivors also displayed tumor formations as presumed causes of death, but we did not study this age period in the current study.3, 3 KCl, 1.3 MgSO4, 2 CaCl2, 2.25 KH2PO4, 8.9 glucose, and 10 sodium pyruvate and was gassed with 95 % O2/5 % CO2 (pH 7.4). The mucosal solution (154 mM NaCl) was gassed with 100 % O2. Basal parameters were measured for 30 min, forskolin (FSK) plus IBMX were then added to the serosal solution with an end concentration of 10 μM plus 100 μM, and JHCO3- was continuously titrated for the subsequent 30 min. JHCO3- was calculated for 5-min intervals.Ussing chamber experiments were performed exactly as described previously . The ser2CO3 solution and isotonic Ringer’s solution to maintain acid/base and fluid balance, which was assessed by periodic blood gas measurements.Mouse anesthesia and control of body functions have recently been described in detail . BrieflyHCO3- in anesthetized mice has been described in detail [HCO3- was determined for each 20-min period, and JHCO3- is expressed as micromoles of base secreted per centimeter of intestine per hour (μM cm−1 h−1). The measurement of fluid absorption was performed as described previously [The surgical procedure for the measurement of duodenal Jn detail , but foreviously . The comMice were sacrificed, and the different intestinal segments were excised, fixed with 4 % paraformaldehyde, embedded in paraffin; tissue sections (2 μm) were prepared, deparaffinized, and stained with hematoxylin and eosin by standard protocols. Antigen Ki67 staining was performed according to the suggestions of the manufacturer .Forskolin and 3-isobuty1-1-methlxanthine (IBMX) were purchased from Alexis Biochemicals . Tetrodotoxin was purchased from Biotrend Chemicals AG . Other reagents were purchased from Sigma-Aldrich  or where specifically mentioned in the “HCO3-” and “Isc” represent the mean value of basal HCO3- secretion and Isc, respectively, “ΔJHCO3-” represents, for each experiment, the maximal response after secretagogue addition averaged over the basal value. The data were analyzed by one-way ANOVA if for multiple comparisons or student’s t test for paired samples. Results were considered significant at P < 0.05.All results were expressed as mean ± SEM, “J- channel, namely CFTR. Slc26a9 was strongly expressed in the stomach, and to some extent the proximal duodenum but not in the more distal GI tract  expression , whereas CFTR KO mice display the highest mortality rate at the time of weaning . To testFigure The additional loss of Slc26a9 strongly increased the mortality in the weaning period. Because the majority of mice disappeared before postnatal day 30, during which the litter was still with the parents, we could not ascertain the cause of death in most instances. However, since the double KO mice were indistinguishable from their littermates at the time of genotyping, we believe with a very high level of confidence that the cause of death was, as in the older mice, intestinal obstruction.3- secretory rates and the transepithelial fluid movements were assessed in the proximal and distal duodenum of anesthetized WT mice. A significantly higher HCO3- secretory rate both in the basal state  and during FSK stimulation  was observed in the proximal (first 1 cm of duodenum) than the distal duodenum (last 1 cm before ligament of Treitz)  Fig.  in vivo.cal Fig. .Fig. 4HC3- secretory rates were also measured in muscle-stripped mucosa of the same segments of WT duodenum in Ussing chamber setups. When studied simultaneously from the same set of mice, proximal duodenal mucosa had a higher basal and FSK-stimulated JHCO3- than the distal mucosa  Fig.  and was num Fig. . In the num Fig. .Fig. 6HCTo search for morphological correlates of the observed functional changes, we performed morphometry of the proximal duodenum in both young (3-6 weeks) and older (above 4 months) Slc26a9 KO and WT mice. The measurement of the duodenal villus and crypt lengths of the proximal duodenum at these two ages revealed, surprisingly, that the crypts and villi were significantly longer in the young Slc26a9 KO than WT mice Fig. , but thaThe proliferative zone was stained with an anti-Ki67 antibody. A longer proliferative zone was observed with intense labeling for Ki67 in many cells, in the duodenum of young mice Fig. . This di−/− and WT mucosa. As described for in vivo experiments [HCO3- but not the FSK-induced ∆JHCO3- was reduced in the proximal duodenum , as well as the rest of the intestine [3- secretory rate and the HCO3- secretory response to FSK were reduced at any age but more so at young age.Because of the marked gradient of Slc26a9 expression along the duodenal length, we studied fluid transport and HCOntestine , 36, 46.3- secretion and electrophysiological parameters in isolated proximal and distal duodenal mucosa of WT and Slc26a9 KO mice, where the submucosal glands are largely removed in the stripping procedure. For these investigations, the small chamber aperture allows us to study the most proximal part of the duodenum, directly after the pylorus. A significant difference in the basal and FSK-induced HCO3- secretory response was again observed between isolated proximal and distal duodenal mucosa. Slc26a9 deletion resulted in a significant decrease in HCO3- secretory response in the proximal duodenum; no difference was observed in the distal duodenum.Fluid movements are dependent not only on epithelial ion transporters but also on systemic influences such as hydration status, perfusion pressure, and acid–base balance. In addition, the submucosal Brunner’s glands  are prob3- conductivity of approximately one eighth of that for Cl- for Slc26a9 expressed in oocytes [3- conductivity of Slc26a9 when expressed in oocytes [3- titration that the ratio of HCO3- to Cl- export into the luminal fluid is between approximately one fifth in the high HCO3--secreting rabbit duodenum [3- secretory flux could in theory be mediated by Slc26a9 itself. If that were the case, we might expect to see an eightfold higher effect of Slc26a9 deletion on the basal Isc, which was not observed. Therefore, we consider it unlikely that the higher HCO3− secretory rate in Slc26a9 WT duodenum is due to the (presumably very low) HCO3− permeability of Slc26a9 itself.Explaining the findings on a molecular level based on currently available data may come up with several different models: Firstly, Loriol et al. reported a HCO oocytes . We knowduodenum  to approduodenum  and unpu− recycling” fashion. This would best explain our observations, but the majority of heterologous expression data as well as our study in the lung do not support this conclusion [Secondly, Slc26a9 could, in theory, be an anion exchanger working in parallel with CFTR, in a classical “Clnclusion , 26–28.3- secretory response in Slc26a9 WT duodenum [sc response to FSK between WT and Slc26a9 KO mucosa, and the fluid secretory response of the duodenum of Slc26 KO appeared normal. Neither parameter accurately reflects CFTR activity in the native intestine, where the rate-limiting steps for anion secretion may be the uptake/generation of the anions [Another frequently discussed molecular model of Slc26-CFTR interactions envisions an interaction of the STAS domain of the Slc26 members with the regulatory domain of CFTR. Bertrand et al. have described a reciprocal stimulation of both Slc26a9 as well as CFTR current by STAS-R domain interaction in a protein kinase A (PKA)-dependent fashion . Ousingsduodenum . Howevere anions , 19, 42.3- secretion but not in Isc response [HCO3- and FSK-induced ∆JHCO3- compared to WT littermates, whereas the distal duodenal mucosa had lower HCO3- secretory rates in DRA-deficient mucosa  Slc26a3 (and possibly Slc26a6) perform this task. It is of note that while Slc26a3 and Slc26a6, as well as CFTR, are expressed at much higher levels than Slc26a9 in the duodenum, their cellular location is not identical for the majority of Slc26a3 and Slc26a6 with that of CFTR and Slc26a9. Since for the STAS-R domain interaction, a 1:1 molar ratio of the transporters is envisioned, it is clear that duodenal CFTR in the crypts does not find abundant Slc26a3 and/or Slc26a6 for interaction. Thus, the Slc26a9-CFTR interaction may be of particular physiological significance in this location. If CFTR preferentially interacts with an anion conductance in the proximal duodenum but with an anion exchanger in the distal duodenum, this may explain why the WT proximal duodenum is in a fluid secretory state and the distal duodenum in a fluid absorptive state. This concept is corroborated by the fact that Slc26a9 deletion results in a fluid absorptive proximal duodenum in young mice.The results resemble those described by Walker et al. in the DRA-deficient duodenum, who found a reduction in basal and FSK-induced HCOresponse . In our osa Fig. . Given tosa Fig. , the dat3− output in the proximal duodenum could be a functional interaction of Slc26a9-mediated Cl− efflux and Slc26a3-mediated Cl− uptake in exchange for HCO3−. However, there is little evidence for coexpression of the two transporters in the same cells in the proximal duodenum. But still, an overlap zone may well exist. The different potential modes of Slc26a9-mediated enhanced anion transport rates in duodenal crypt cells are depicted in the cartoons displayed in Fig. Another possibility to explain the higher basal HCOi, which caused alterations in the WNT/β-catenin signaling [i and cell volume, thus having an inhibitory effect on proliferation [A surprising finding of this study was the enhanced proliferative status of the proximal duodenum in the young Slc26a9 KO mice, with lengthening of both the crypts and the villi. A similar observation has been previously made in CFTR-deficient duodenum . The estignaling . We currferation .The Slc26a9 KO mice develop hypergastrinemia with ageing. We did find not find evidence in the literature that hypergastrinemia cause duodenal crypt and villus elongation, or that duodenal crypt cells possess the CCK-B/gastrin receptor, or that they are more sensitive to gastrin at young age of the mice, or that the effect of gastrin in the duodenum is restricted to the proximal duodenum. In addition, KCNQ1 KO mice also had massive hypergastrinemia and a loss of acid secretory capacity from birth  but no dSlc26a9 gene? Our data clearly show that Slc26a9 expression is negligible in the part of the murine and human intestine that is obstructed in the murine CFTR-deficiency-related intestinal obstruction (mid jejunum to ileum) and human CF meconium ileus ; and Slc26a9 expression is not increased in CFTR deficient compared to WT distal small intestine and colon (data not shown). Nevertheless, the additional deletion of Slc26a9 in CFTR KO mice caused a massive decrease in survival during and after the weaning phase, whereas Slc26a9 KO mice had a high perinatal death rate, most likely due to pulmonary complications [How do we reconcile our findings of altered ionic transport in the proximal duodenum of Slc26a9 KO mice with an increased incidence of meconium ileus in CF infants having polymorphisms in the ications , but hadSlc26a9-deficient mice had a virtually absent HCO3- secretory response to luminal acid, while that to FSK was intact [3- secretion only partially overlap at the level of the epithelium [3- secretory response is mediated by a vagal reflex whereas the FSK-induced HCO3- secretory response is not affected by vagotomy [We therefore believe that the reason for increased intestinal obstructions in CF mice or humans with the absence or malfunction of Slc26a9 is the upper GI tract dysfunction resulting in maldigestion and impaired downstream signaling. The severe gastric phenotype has been previously described . Anothers intact . That stithelium , and anovagotomy . Firstly3- secretion and fluid transport, particularly at young age. Slc26a9 deletion strongly reduces the survival of CFTR KO mice, particularly in the weaning phase, where the mice die of intestinal obstruction. Slc26a9 is not only expressed in the stomach and proximal duodenum in mouse and humans, but also to some extent in the pancreas, at similar expression levels to those in the distal duodenum. It is not expressed at significant levels in those intestinal segments that actually experience the obstructions . Our data suggest that the higher risk of meconium ileus in CF infants with Slc26a9 polymorphisms is due to upper intestinal dysfunction including the stomach, the duodenum, and possibly the pancreas. We do not yet know the cellular location of pancreatic and biliary tract Slc26a9, but if it is in the ductal epithelia, the effect on biliary and pancreatic HCO3- secretion may be similar to that in the duodenum. Future studies in biliary and pancreatic secretion of Slc26a9-deficient mice may add to our understanding why polymorphisms in this gene are risk factors not only for meconium ileus in CF infants, but also for CF diabetes.In summary, our study shows that even a low Slc26a9 expression level can cause marked decrease in epithelial HCOESM 1(DOC 261 kb)Below is the link to the electronic supplementary material.


Camels infected with MERS-CoV show few symptoms and likely transmit the virus to humans and other camels through respiratory secretions. In 2012, a novel coronavirus associated with severe respiratory disease in humans emerged in the Middle East. Epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of Middle East respiratory syndrome coronavirus (MERS-CoV). Here we provide experimental support for camels as a reservoir for MERS-CoV. We inoculated 3 adult camels with a human isolate of MERS-CoV and a transient, primarily upper respiratory tract infection developed in each of the 3 animals. Clinical signs of the MERS-CoV infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. We detected infectious virus in nasal secretions through 7 days postinoculation, and viral RNA up to 35 days postinoculation. The pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-to-human transmission. The Middle East respiratory syndrome coronavirus (MERS-CoV) was first recognized in 2012 related to a fatal human case of pneumonia in Saudi Arabia  was provided by the Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands. The virus was propagated in Vero E6 cells cultured in Dulbecco modified Eagle medium  supplemented with 2% fetal bovine serum, 2 mmol/L glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin.Camelus dromedarius) were obtained through private sale; the animals tested negative by neutralization assay for MERS-CoV and for bovine coronavirus by ELISA. Camels 1, 2, and 3 were 2, 3, and 5 years old, respectively. Camels 1 and 2 were intact males, and camel 3 had been castrated. Animals were housed in an Animal Biosafety Level 3 facility for the duration of the experiment and fed ad libitum. Camels were acclimated to the facility for 2 weeks before virus inoculation. We sedated the camels with xylazine, then inoculated them with a total dose of 107 50% tissue culture infective dose (TCID50) of MERS-CoV (strain HCoV-EMC/2012) in a total volume of 15 mL, by way of intratracheal (8 mL using transcutaneous catheter), intranasal (3.3 mL in each nostril by expulsion from a syringe), and conjunctival  routes. The routes of inoculation and infectious dose were chosen to reflect a combination of most likely routes of exposure and to increase the potential of infection. The animals were observed at least 1× daily for the duration of the experiment for behavior, food consumption, activity level, and nasal discharge. Rectal temperature was taken daily from 2 to 7 days postinoculation, then 3× weekly until the animals were euthanized. Nasal and oral swab samples and fecal samples were collected into virus transport medium or virus lysis buffer daily from 0 to 7 days postinoculation (dpi), then 3× weekly until the animal was euthanized. Blood was collected into evacuated EDTA and serum-separating tubes daily at 0–7 dpi and 3× weekly thereafter. Urine was collected by convenience and at necropsy. To evaluate whether virus is exhaled from infected camels, a funnel was placed over the muzzle of each camel and connected to a vacuum pump to capture exhaled air in tissue culture media  with an All Glass Impinger . Exhaled breath was collected for ≈2 minutes and analyzed by quantitative real-time PCR (qPCR) and virus titration. On days 5, 28, and 42, camels 1, 2, and 3, respectively, were euthanized, and samples were collected from nasal turbinates, lungs, trachea, larynx, pharynx, liver, spleen, kidney, bladder, urine, duodenum, jejunum, colon, rectum, abomasum, forestomachs, prescapular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, mesenteric lymph node, medulla, and olfactory cortex.Three native-born adult male dromedary camels  according to the manufacturer’s instructions. For detection of viral RNA, we used 5 μL of RNA in a one-step real-time reverse transcription PCR upE assay  for MERS-CoV virus by plaque assay. Briefly, 10-fold serial dilutions of samples were prepared in BA-1 medium  containing 100 mg gentamicin, 200,000 U penicillin G, 100 mg streptomycin, and 5 mg amphotericin/L; plaque assay was conducted as described for West Nile virus  as a primary antibody.<2 weeks. The nasal discharge drained from both nares and varied in character from serous to purulent; minor hemorrhage was observed on some occasions, but may have been caused by trauma that occurred during collection of samples.Each camel showed minor clinical signs of disease, consisting of rhinorrhea , panel A1.2 and 101.4 TCID50 equivalent/mL) at 3 and 5 dpi, but infectious virus was not detected.MERS-CoV shedding started during 1–2 dpi, as detected by the presence of infectious virus and viral RNA by qPCR in nasal swab samples. Infectious virus shedding was detected <7 dpi, and shedding of viral RNA was detected <35 dpi in nasal swab samples . Low conInfectious virus was detected in tissues from camel 1, which was euthanized on 5 dpi, but not in tissues obtained from camels 2 and 3, which were euthanized at28 and 42 dpi, respectively. Infectious virus was detected in tissues of the upper respiratory tract (URT), including nasal turbinates, olfactory epithelium, pharynx, and larynx. In the lower respiratory tract, infectious virus was detected in the trachea and in 1 of 4 lung lobes tested. Infectious virus was also detected in the retropharyngeal, mediastinal, mesenteric, and tracheobronchial lymph nodes . On necrSerum samples were collected weekly from the camels to monitor the generation of neutralizing antibodies specific to MERS CoV. Each of the 3 camels was seronegative before inoculation. Robust MERS-CoV specific antibody responses developed in camels 2 and 3 , detected first on 14 dpi with a plaque-reduction neutralization test titer from 20 to 40 that increased to 640 at 35 dpi . Camel 1Epidemiologic and surveillance data on the emergence of MERS-CoV strongly point toward a role for dromedary camels as a reservoir for zoonotic transmission (The large quantities of MERS-CoV shed in nasal secretions by each of the 3 camels suggest that camel-to-camel and camel-to-human transmission may occur readily through direct contact and large droplet, or possibly fomite transmission. Histopathologic examination revealed that the URT, specifically the respiratory epithelium in the nasal turbinates, is the predominant site of MERS-CoV replication in camels.Neutralizing antibodies were detected from 14 dpi onward, reaching a maximum neutralizing titer of 640 after 35 days. Serologic studies in camels in the field have reported MERS-CoV neutralizing titers as high as 5,120 (The study reported here was done on the basis of inoculation of 3 male animals with a human isolate of MERS-CoV, and the study design we used imposed several limitations on how these data inform what occurs in natural infections. The camels we inoculated were exposed to a high dose of virus by 3 simultaneous routes of inoculation. In retrospect, the inoculation dose does not seem excessive, based on the large quantity of virus shed nasally in all 3 animals . The tot


Aquaparamyxovirus and Ferlavirus, to accommodate this increasing diversity, several recently isolated or characterized viruses remain unclassified beyond the subfamily level. In the present study, among 985 bats belonging to 6 species sampled in the Belinga caves of Gabon, RNA of an unclassified paramyxovirus  was discovered in 14 African sheath-tailed bats (Coleura afra), one of which exhibited several hemorrhagic lesions at necropsy, and viral sequence was obtained in two animals. Phylogenetically, BelPV is related to J virus and Beilong virus (BeiPV), two other unclassified paramyxoviruses isolated from rodents. In the diseased BelPV-infected C. afra individual, high viral load was detected in the heart, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by J virus. BelPV was not detected in other tested bat species sharing the same roosting sites and living in very close proximity with C. afra in the two caves sampled, suggesting that this virus may be host-specific for C. afra. The mode of transmission of this paramyxovirus in bat populations remains to be discovered.Bats are known to harbor multiple paramyxoviruses. Despite the creation of two new genera,  Paramyxovirinae and Pneumovirinae. Paramyxovirinae has recently been subdivided into seven genera: Aquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Morbillivirus, Respirovirus, and Rubulavirus (http://ictvonline.org/virusTaxonomy.asp?version=2012). Viruses of this family affect a wide range of animals, including primates, birds, carnivores, ungulates, snakes, cetaceans and humans, and cause a wide variety of infections, such as measles, mumps, pneumonia and encephalitis in humans, and distemper, peste des petits ruminants, Newcastle disease and respiratory tract infections in animals. However, several paramyxoviruses (PVs) have not been classified into any of these seven genera, including Nariva virus (NarPV) Mossman virus (MosPV) Beilong virus (BeiPV) J virus (JPV) Tupaia paramyxovirus (TupPV) Tailam virusSalem virus isolated from horses Members of the Paramyxoviridae family are pleomorphic enveloped viruses Bats have a close evolutionary relationship with several genera of mammalian paramyxoviruses Coleura afra) As part of a large-scale investigation of viral diversity in bats and of associated zoonotic risks, we have previously detected a bat paramyxovirus in one insectivorous African sheath-tailed bat  The study was conducted in the Belinga mountains (northeast Gabon), where Ebola outbreaks occurred in 1994–1996 and 2001–2002. All the capture events, animal handling, euthanasia and transfer of samples across country borders were performed in accordance with the guidelines of the American Society of Mammalogists  was carried out with authorization from the Gabonese Ministry of Water and Forestry (Département de la Faune et de la Chasse – Authorization N°003/MEFEPA/SG/DGEF/DFC and N°0021/MEFEPA/SG/DGEF/DFC) and the Gabonese Ministry of Higher Education, Scientific Research and Innovation .Samples of liver, spleen, kidney, lung, heart, gut, brain and salivary glands were collected, stored in liquid nitrogen and transferred to the CIRMF laboratory , where they were stored at −80°C until analysis. Blood samples were also collected, except from the smallest individuals (body mass <12 g). A total of 985 bats  were samC. afra out of the 26 individuals, from Batouala cave, exhibited diarrhea and severe hemorrhagic lesions in both thoracic and abdominal organs, along with lung congestion and pleurisy.Interestingly, at autopsy, only one Virological screening of the diseased bat was performed to identify the cause of these lesions. The screening process included the whole Paramyxoviridae family and also filoviruses [Marburg virus (MARV) and Zaire Ebola virus (EBOV)], given the nature of the lesions and the known filovirus tropism for bats. Briefly, approximately 100 mg each of this animal's liver and spleen were pooled and crushed in 600 µl of cold PBS in a ball-mill tissue grinder . Total RNA was extracted using a Biorobot EZ1 and the EZ1 RNA tissue mini kit (Qiagen) according to the manufacturer's guidelines. The RNA was then tested for paramyxoviruses, using three heminested reverse transcription-PCR (hnRT-PCR) assays targeting the polymerase gene 5′-GGCGGCTCTTAAAAGTGAATG-3′; GB09-478-R, 5′-GCGGGGTCAAATTGGTCAT-3′; probe: GB09-478-P, 5′-TCCAGCACAAACATATCCGAGAAGGCTAG-3′) was designed within the initial PCR fragment and was used to test total RNA extracted from mixed liver and spleen samples from each of all the other bat species. The amplification was performed in a final volume of 25 µl, containing, 12.5 µl TaqManR 2X PCR Master Mix (Applied Biosystems), 0.5 µl each primer and probe (10 µM), 1 µl bovine serum albumin (1 µg/µl) (Invitrogen), 5 µl cDNA and RNAse-free water (Invitrogen). Amplification generally involved 2 min at 55°C, 10 min at 95°C followed by 45 cycles of 15s at 95°C and 1 min at 58°C.To further investigate the presence of the virus in bat populations, a strain-specific real-time RT-PCR assay  trapped in the Faucon and Zadié caves between November and December 2009, June 2010, and January, March and April 2011. After the morphologic species determination, insects were crushed by monospecific pools  in 300 µl PBS. Total RNA was extracted using 100 µl of the supernatant from each pool by using the RNeasy Mini Kit (Qiagen) and then was tested with the specific real-time RT-PCR assay described above.Transmission by hematophagous arthropods was studied. Hematophagous arthropods were collected inside Faucon and Zadié caves. Mosquitoes were sampled using CDC light traps whereas bat-flies were manually collected on bats  assays yielded an amplicon of 559 base pairs (bp) from diseased bat, in collaborative work with the Bonn Institute of Virology Bonn (Germany). The PCR product was sequenced with dye terminator chemistry (Applied Biosystems). No other virus was detected in the diseased bat.Morbillivirus and Henipavirus , belonged to the Paramyxoviridae family. It showed 65% and 66% nucleotide identity, respectively, with JPV and BeiPV, and had pairwise nucleotide identities of 36–42% with the Henipavirus group and 37–40% with the Morbillivirus group. Phylogenetic analysis indicated that this bat PV, that we named Belinga bat virus (BelPV), clustered with unclassified paramyxoviruses and was located between the genera ipavirus .C. afra bats, including the diseased animal, were positive. Bat samples positive by specific real-time RT-PCR were then tested with RMH-hemi nested-PCR for molecular characterization. Another 439-bp PCR product was amplified from one liver-spleen pool from a second C. afra specimen, and was sequenced. These two phylogenetically related sequences  (BelPV-specific RNA was detected only in a caves) . Interes2 = 2.476, df = 1, p>0.20). In the bat exhibiting severe hemorrhagic lesions (GB09 478), both the liver and heart were BelPV-positive, with a higher viral load in the heart .BelPV RNA was detected in the heart (8/12) and liver (5/14), at low and variable loads . No signIn total, 432 arthropods were collected inside Faucon and Zadié caves, including Culicidae (10 Uranotaenia nigromaculata and 320 Culex wigglesworthi), Nycteribiidae  and Streblidea  . No arthHenipavirus and Morbillivirus. The same phylogenetic position had been observed with MosPV and J-V The BelPV nucleotide sequence obtained showed similarity with the JPV and BeiPV sequences. BelPV has previously been reported to hold a phylogenetic position between the genera Eidolon helvum (megachiroptera bat) in Africa In this study, organs with high BelPV concentrations are different from those found with high paramyxovirus concentrations in pteropodids and microchiroptera bats. In microchiroptera bats from Brazil, spleen has been found more positive than the others organs with highest viral load, as in The within-host BelPV distribution tended to be organ-specific. BelPV seemed to be restricted to the heart and liver. In contrast, JPV has been isolated from blood, lung, liver, kidney and spleen of experimentally infected laboratory mice Coleura afra and not in other bat species sharing the same roosting sites and living in very close proximity in the two caves sampled. However, it has been shown that bats of different species occupying the same roosting sites can share the same viruses. Marburg virus had been detected in Rousettus aegyptiacus and Hipposideros sp. bats living in Kitaka cave in Uganda Miniopterus inflatus and Rousettus aegyptiacus bats caught in Goroumbwa mine in the Democratic Republic of the Congo C. afra, as well as restricted intraspecies transmission. Henipaviruses occur naturally in fruit bats belonging to the genus PteropusRhinolophus bats We detected BelPV only in C. afra. Indeed, high viral load was detected in the heart of the diseased bat, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by JPV In view of our data we can assume that BelPV might have pathogenic potential for its host Geomyces destructans, causative agent of white-nose syndrome, may become pathogenic in hibernating bats in North America Coleura afra species are considered in order to find BelPV again for further studies . However, Coleura afra is a migratory species living in colonies of several hundred individuals. In Gabon, this species, which has been recently described, is not present all year round in the caves of the north-east of the country, making the studies on this species difficult and thereof partly explaining the lack of virological studies.Although BelPV RNA was also detected in asymptomatic bats, pathogenicity may appear in long term under some immunological and/or ecological conditions. Indeed, virus must not induce pathology to persist or adapt within its reservoir host. Many authors suggested that persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population Some viruses appear to cause clinical disease in wild-living bats; these include lyssaviruses and an ebola-like filovirus named Lloviu virus Bats are the natural reservoirs for many viruses, including emerging zoonotic viruses such as SARS-CoV C. afra cannot be ruled out. Indeed, a haemosporidian parasite (Polychromophilus) was found in a blood parasite vector (Penicillidia fulvida) in Faucon cave in Gabon in 1977 M. inflatus (greater long-fingered bat) from the same cave in 2010 and 2011 All the blood-sucking arthropods collected from bats, as well as mosquitoes collected in the caves where bat sampling took place, were negative for BelPV, in keeping with the lack of known PV vectors C. afra and BelPV could serve as an interesting model, (i) to evaluate modes of transmission within host populations, (ii) to study host-virus interactions (pathogenesis and host specificity), and (iii) to evaluate the zoonotic risk of a newly identified virus.This association between C. afra populations and a broader diversity of arthropod vectors, spanning larger areas and time scales, are needed to confirm this apparent host-virus specificity, and to determine the modes of BelPV transmission. Further studies are needed to characterize complete BelPV genome and demonstrate the pathogenicity of this virus for its host Coleura afra.Further studies of


Viruses cause epidemics on all major cultures of agronomic importance, representing a serious threat to global food security. As strict intracellular pathogens, they cannot be controlled chemically and prophylactic measures consist mainly in the destruction of infected plants and excessive pesticide applications to limit the population of vector organisms. A powerful alternative frequently employed in agriculture relies on the use of crop genetic resistances, approach that depends on mechanisms governing plant–virus interactions. Hence, knowledge related to the molecular bases of viral infections and crop resistances is key to face viral attacks in fields. Over the past 80 years, great advances have been made on our understanding of plant immunity against viruses. Although most of the known natural resistance genes have long been dominant R genes (encoding NBS-LRR proteins), a vast number of crop recessive resistance genes were cloned in the last decade, emphasizing another evolutive strategy to block viruses. In addition, the discovery of RNA interference pathways highlighted a very efficient antiviral system targeting the infectious agent at the nucleic acid level. Insidiously, plant viruses evolve and often acquire the ability to overcome the resistances employed by breeders. The development of efficient and durable resistances able to withstand the extreme genetic plasticity of viruses therefore represents a major challenge for the coming years. This review aims at describing some of the most devastating diseases caused by viruses on crops and summarizes current knowledge about plant–virus interactions, focusing on resistance mechanisms that prevent or limit viral infection in plants. In addition, I will discuss the current outcomes of the actions employed to control viral diseases in fields and the future investigations that need to be undertaken to develop sustainable broad-spectrum crop resistances against viruses. As obligatory intracellular parasites, plant viruses depend on the host machinery to multiply and invade their hosts. In their simplest form, viruses consist of a DNA or RNA genomic segment encoding only few genes and encapsided into a protein shell, called the capsid. Because of natural physical barriers , viruses are delivered into plant cells through wounds or through the action of vectors  that feed on or infect the plants. Following entry into a host cell and genome decapsidation, the infectious cycle includes translation and replication of the viral genome, assembly of progeny virus particles, generalized invasion of the host through cell-to-cell and long-distance movements of viral particles or ribonucleoprotein complexes and finally, transmission to new hosts by vectors. In some cases, transmission to the following generation of host plant is also observed as a result of seed infection. In 2012, the International Committee on Taxonomy of Viruses reported 92 genera of plant viruses of which 82 were assigned in 21 different families .Plant infection by viruses causes physiological disorders responsible for plant diseases of economic and agronomic significance in many crops. Widely employed in fields, the use of plant varieties carrying genetic resistances constitutes the most effective, economical and ecological measure to control viral infections. The last decades have seen substantial advances on the molecular dialog between viral pathogens and their plant hosts, bringing new strategies directly exploitable in crop improvement programs. Nevertheless, the spread of crop viral pests has increased dramatically in recent years. Globalization, trade and climate change, as well as reduced resilience in production systems due to decades of agricultural intensification have all played a part.This review intends: (i) to provide a brief overview of severe virus-associated plant diseases and their impact on crop production, (ii) to summarize prophylactic strategies employed to control viral epidemics in fields, (iii) to bring an update on current knowledge about plant resistances against viruses, (iv) to present the technical approaches currently employed in crop improvement programs, and (v) to discuss how antiviral mechanisms based on PAMP-triggered immunity (PTI) could be the source of novel plant resistances in fields.Management of plant virus diseases is a matter of vital importance and concern to farmers, horticulturists, foresters, manufacturers, as well as consumers. It is well-established that virus diseases in different crops cause enormous losses all over the world in terms of quantity and/or quality of products. Although it’s very difficult to put a clear figure on the financial impact of plant viruses in agriculture, the yield losses that can be ascribed to plant viruses are estimated to cost worldwide more than $30 billion annually .Cassava mosaic begomoviruses cause more than 25 million tons of losses per year in Africa, India, and Sri Lanka  were estimated to over 100 million trees worldwide ] since the 1970s have exceeded 10 billion euros .Because viral agents are obligate intracellular parasites, curative treatments of virus infections are impossible, making viral diseases very difficult to control in fields. Prophylactic control measures are therefore crucial in combating epidemics on crops. They consist mainly on combining cultural practices, biosecurity measures and organism-vector management  for their plant-to-plant spread. Hence, viral diseases can be efficiently controlled by limiting the populations of their vectors with the applications of appropriate pesticides. The use of non-host “trap plants” may be also considered to attract vectors to reduce the number of individuals feeding on the crop of interest and thus, the transmission of the disease .Epidemics often arise from new viruses or new variants of classic viruses that spilled over from reservoir species to crops. Although this phenomenon results from a complex evolutionary process in which the main players are ecological factors, virus genetic plasticity and host factors, viral diseases can be controlled by managing the spatial structure and composition of field parcels, which impacts resistance durability .Minimizing viral epidemics involves the respect of international legislations concerning worldwide trade of virus-free plant material, which applies to any development stage of a plant that can be carrier of viruses , as well as manipulation of decontaminated horticultural tools.The use of genetically resistant plants is one of the most efficient, sustainable and frequently employed strategies to control virus infections in fields. For centuries, it has involved plants selected by breeders for their agronomic proprieties combined to the absence of disease symptoms. However, from the middle of the 20th century, plant improvement programs capitalize strongly on the knowledge associated to plant–virus interactions to develop resistant varieties exploitable in agriculture.Figure 2).Faced with viral attacks, plants defend themselves through several resistance layers, that are complementary in terms of defense timing (at early or late infection steps), location (in the first infected leaf or in systemic tissues) and targeting the virus-derived molecules  identified in plant–virus interactions belong to the nucleotide binding site-leucine-rich repeat (NBS-LRR) class, that specifically recognize the viral avirulence (avr) gene products, through the establishment of the so-called “gene-for-gene” interaction. Although a direct physical interaction between the avr and R gene products had originally been suggested, the current understanding favors the more sophisticated “guard hypothesis” model  through the recognition of the PVX capsid (CP). Rx1 CC domain has been shown to form a heterodimer with the cellular ranGTPase-activating protein 2 (ranGAP2), interaction required for Rx1 function , in an ATP dependent manner , HSP90 (Heat shock protein), and RAR1 , a phenomenon involving the programmed death of the infected and neighboring cells, with the consequences to confine the pathogen in the hypersensitive lesion and to prevent any further pathogen spread in the plant. A functional module that mediates HR against viruses  requires the interaction of two lipases, EDS1 (Enhanced disease susceptibility 1) and PAD4  with the protein SAG101 , a mechanism that has been demonstrated for both N and Rx1 genes  production, salicylic acid (SA) accumulation, and extensive transcriptional reprogramming. In addition, the activation of rmovirus . Recent rmovirus . Meanwhix1 genes  and thatR genes, the RTM genes (for Restricted TEV Movement) were the first cloned non-NBS-LRR dominant resistance genes against viruses. Genetic characterization of natural Arabidopsis accessions and mutants showed that at least five dominant RTM genes are involved in resistance to Tobacco etch potyvirus (TEV), Lettuce mosaic potyvirus (LMV), and PPV , the translation elongation factors 1A and 1B (eEF1A and eEF1B) and the poly(A)-binding proteins (PABPs) 2, 4, and 8  belonging to the eIF4E and eIF4G families  protein in the recessive resistance to bymoviruses (ra blocking vascular transport of Potato potyvirus A (PVA) was genetically characterized in potato  contributing to systemic resistance against PPV  has been recognized as an evolutionarily conserved process in most eukaryotes, that is triggered by double-stranded RNAs (dsRNAs). These dsRNAs are processed by ribonuclease III-type DICER-like (DCL) enzymes into small RNAs (sRNAs), 21–24 nucleotides in length, that are incorporated into an RNA-induced cytoplasmic silencing complex (RISC), whose key catalytic component corresponds to one member of the ARGONAUTE (AGO) protein family. Once integrated into the RISC, sRNAs base-pair to their target-mRNA and induce their cleavage . In planFigure 2C). What initially appeared counter-intuitive, infection from DNA viruses produce also dsRNAs, most likely via bidirectional convergent transcription  during the replication step. These molecules are targeted by the RNA-silencing machinery to produce virus-derived small RNAs  encoding a methyltransferase that protects siRNA and miRNA duplexes from degradation (Ty-1 and Ty-3 conferring resistance to Tomato yellow leaf curl begomovirus (TYLCV) has been recently shown to encode a tomato RDR. Although resistant plants do not show symptoms upon a challenge with TYLCV, low levels of virus are still detectable, a phenomenon characteristic of a virus tolerance more than a real resistance . In the case of TMV-triggered HR, the response persists up to 3 weeks during the time plants are protected against not only TMV but also other pathogens (N. benthamiana (During the R-mediated resistance activation (see the Section “Dominant Resistances” in this review), cellular responses elicited at the infection site are emitted to distant non-infected tissues, resulting in a resistance or in reduced susceptibility state that can remain efficient during several weeks . This phathogens . How SARathogens . Moreoveathogens . The sigathogens . Recent athogens . Jasmoniathogens  but is ethamiana . It is lthamiana . Interesthamiana . Althougthamiana , ethylenthamiana , and brathamiana .Ultimately, the main objective of research on plant–virus interactions consists on the implementation of efficient antiviral resistances in crop plants. For long, genetics-mediated resistance strategies have involved exploiting plant natural variability by introgression of resistance genes through a classical breeding process. However, the onset of evolved pathogens able to overcome these resistance, sometimes very rapidly after resistance deployment , raised de novo whole genome shotgun (WGS) sequencing of reference genotypes and whole genome resequencing (WGRS) of several cultivars, land races, and wild relatives , restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSRs) contributed, through what is called “Marker-assisted selection (MAS),” to make substantially easier, quicker, and more accurate the selection of resistant genotypes during the introgression steps . Importaelatives . Despiteelatives , MAS hasPotato potyvirus Y . Initially reported in transgenic tobacco plants expressing the TMV CP  plants resistant to viral attacks are grouped into three major categories. First of all, resistance genes with a dominant determinism can be introduced to another plant species. The best examples in plant–virus interactions involve the successful transfer of d plants  but it sd plants . The oppe TMV CP , the rese TMV CP . These oe TMV CP . In the e TMV CP . In spitA. thaliana, both TILLING and ecoTILLING have spread rapidly to other model plants  and major crops . It seems now clear that these strategies are emerging as major crop improvement tools, with especially successful examples of recent applications to antiviral protection  method, that consists of a classical mutagenesis step followed by the targeted search for plants carrying a mutation in a gene of interest . By creaotection .S-methyl, that leads to plant protection against many pathogens including viruses  is able to infect a broad range of herbaceous crops  and fruit trees  without causing any symptoms, this virus was identified as an excellent candidate for VIGS-mediated vaccination, that could be simply applied widely by replacing the insert with a sequence derived from a different virus. This strategy has been recently illustrated , and that triggers virus-induced gene silencing (VIGS) targeting both primary and challenge viruses . Primaryin Japan . Moreovein Japan . Howeverin Japan . Given Austrated  and reprIn an attempt to increase resistance durability in fields, crop improvement requires a continuous pipeline of new resistance genes. Given the importance of PAMP-triggered immunity (PTI) in the field of pathology over the last decades, PTI against plant viruses represents an unexplored question that needs to be addressed.Over the last decades, a concept revolutionizing the understanding of immunity emerged in plant pathology. This new concept stems from the ability for each organism to discriminate between self and non-self molecules through the action of pattern recognition receptors (PRRs) perceiving specific microbial molecular signatures, named pathogen-associated molecular patterns (PAMPs). Perception of PAMPs by these immune receptors induces a downstream signaling cascade including PRR association with positive regulators, phosphorylation events, successive activation of cytoplasmic kinases (including the MAP kinases) and defense-related transcription factors, as well as specific defense genes expression. This range of fast, efficient and multi-layered defense responses is referred as PTI . To counTo date, most current models about plant innate immunity exclude viruses, as they are not generally viewed as encoding typical PAMPs and effectors that would trigger PTI and ETI responses . The facR gene-based immunity in plant–virus interactions involves specific recognition of viral avirulence factors by host-encoded NBS-LRR proteins and leads to HR and SAR (described previously in this review), recent hypotheses advance that it may correspond to antiviral ETI mechanisms  and MDA5 (Melanoma differentiation-associated gene 5), both highly activated by dsRNAs, as well as 5′ triphosphate-RNAs for RIG-I , a family of a dozen type I-transmembrane proteins containing a domain with LRRs and a tail that contains a conserved region called Toll/IL-1 receptor (TIR) domain , and shaor RIG-I . In the or RIG-I .Bean pod mottle comovirus (Arabidopsis resistance to plant viruses (Arabidopsis defense against the Cabbage leaf curl geminivirus (CaLCuV). Strikingly, NIK1 is cleaved by the viral protein NSP during the infection, suppressing its kinase activity and thus enhancing susceptibility to CaLCuV infection  colonization strategies employed by plant and animal viruses inside host cells are highly similar , (ii) plFigure 3), that does not invalidate previous models but instead tries (i) to bring the concept of PTI as a novel plant antiviral mechanism, and (ii) to integrate the concept of innate (PTI and ETI) and adaptive (gene silencing) antiviral immunities, both triggered by similar virus-derived elicitor molecules, in order to address the important question of plant defense against viruses in a more holistic way.Thus, strongly based on the understanding of plant immune pathways and the knowledge on antiviral mechanisms from the animal field, I propose here a model of plant immunity against viruses . Successful viruses encode specialized effectors that are able to suppress this defense layer. In turn, specific R gene products interact with these effectors (playing then the role of avirulence factors), leading to ETI. In the case of RNAi-based adaptive immunity, the viral elicitor molecules (mostly dsRNAs) are recognized by DCLs. This leads to virus degradation at the nucleic acid level, a process that can be suppressed by a second class of virus-encoded effectors, commonly named silencing suppressors. Interestingly, Thus, following the virus entry into a plant cell, viral nucleic acids are perceived as PAMPs by specific intracellular PRRs triggering PTI pathways  are highly conserved within virus families and differ radically from prokaryotic or eukaryotic nucleic acids. It is these typical viral features that are perceived by specialized PRRs in animals , triggering very fast and very efficient immune responses. Because the innate pathway is solely programmed by structural and nucleotide sequence genomic features, plant antiviral PTI should be a remarkably versatile mechanism because it could respond virtually to any plant virus. Moreover, timeliness of defense responses is a key parameter in resistance success, especially in the case of viruses, given their tremendous adaptation abilities. And yet, the first measurable PTI responses are within minutes after PAMP perception , making In the world agriculture context, intensification of cultural practices, climate alterations, and extensive exchanges affecting global markets are associated with an increased incidence of plant viral diseases. In consequence, management of viral epidemics is a matter of global food security. Nowadays, antiviral measures employed in fields benefits greatly from the latest knowledge highlighted by plant virologists, geneticists and molecular biologists, as well as engineers and breeders, whose coordinated effort enables a deployment in crop improvement programs. This notwithstanding, the situation in fields remains worrying, given the emergence of numerous crop diseases ascribed to viruses, a statement that could be aggravated in the future through climate changes that might affect strongly virus and organism-vectors populations in terms of magnitude, locations and dynamics . In a coThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.