Hand disinfectants are important for the prevention of virus transmission in the health care system and environment. The development of broad antiviral spectrum hand disinfectants with activity against enveloped and non-enveloped viruses is limited due to a small number of permissible active ingredients able to inactivate viruses.A new hand disinfectant was developed based upon 69.39 % w/w ethanol and 3.69 % w/w 2-propanol. Different amounts of citric acid and urea were added in order to create a virucidal claim against poliovirus (PV), adenovirus type 5 (AdV) and polyomavirus SV40 (SV40) as non-enveloped test viruses in the presence of fetal calf serum (FCS) as soil load. The exposure time was fixed to 60 s.10 reduction of viral titers. Furthermore, this formulation was able to inactivate PV, AdV, SV40 and murine norovirus (MNV) in quantitative suspension assays according to German and European Guidelines within 60 s creating a virucidal claim. For inactivation of vaccinia virus and bovine viral diarrhea virus 15 s exposure time were needed to demonstrate a 4 log10 reduction resulting in a claim against enveloped viruses. Additionally, it is the first hand disinfectant passing a carrier test with AdV and MNV.With the addition of 2.0 % citric acid and 2.0 % urea an activity against the three test viruses was achieved demonstrating a four logIn conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in current guidelines and thereby contributing as valuable addition to the hand disinfection portfolio. Virus transfer via human hands is a major route of infection inside and outside medical settings . The eff10 titer reduction factor (RF of 4) against vaccinia virus and bovine viral diarrhea virus (BVDV) are active against all enveloped viruses  [Worldwide there are differences regarding the number of test viruses needed to be inactivated in standardized tests before a broad spectrum claim including non-enveloped viruses can be given by manufacturers for a hand disinfectant . Regulatrucidal) , 24. Disrucidal) . The disrucidal) . In 2012rucidal)  and the rucidal) .Table 1OMost biocidal active substances used in hand hygiene have no difficulty inactivating enveloped viruses , which aTo date all of the products that claim virucidal activity for hand hygiene under German DVV/RKI Guideline are alcohol-based formulations containing either high amounts of ethanol or an ethanol/1-propanol mixture supplemented with phosphoric acid . Yet higThe poliovirus type 1 strain LSc-2ab (Chiron-Behring) was obtained from PD Dr. O. Thraenhart, Eurovir, D-14943 Luckenwalde. The adenovirus type 5 strain Adenoid 75 was obtained from PD Dr. A. Heim, Institute of Medical Virology, Hannover Medical School, D-30625 Hannover. Vaccinia virus strain Elstree  originated from the Institute of Medical Virology and Immunology of the University of Essen, D-5122 Essen. Polyomavirus SV40 strain 777 was obtained from PD Dr. A. Sauerbrei, Institute of Virology and Antiviral Chemotherapy at the Friedrich Schiller University of Jena. Murine norovirus S99 (MNV) originated from PD Dr. E. Schreier, Head of FG15 Molecular Epidemiology of Viral Pathogens at the Robert Koch-Institute (RKI) in D-13302 Berlin and BVDV strain NADL (VR-534) was obtained from Dr. S. Bendtfeld, Institute of Virology at the School of Veterinary Medicine Hannover (Tierärztliche Hochschule), D-30559 Hannover.7 to 109 TCID50/mL. Poliovirus was propagated in BGM cells  and adenovirus in A549 cells . The A549 cells originated from the Institute of Medical Virology, Hannover Medical School. Vaccinia virus replication was performed in Vero cells (monkey kidney cell line) obtained from Vircell, SL in ES-18329 Santa Fe, Spain . Polyomavirus SV40 was propagated in CV-1 cells (kidney cells of African green monkey) and MVM in A9 cells . MNV was propagated in RAW 264.7 cells . EKL cells  for BVDV propagation were used. These cells originated from Mrs. A. Kyas . Poliovirus and MNV were replicated in Dulbecco’s Modified Eagle’s Medium (DMEM), all other viruses in Eagle’s Minimum Essential Medium with Earle’s BSS (EMEM).The test virus suspensions were prepared by infecting monolayers of the respective cell lines. The virus titers of these suspensions ranged from 10The formulations were supplied by Oro Clean Chemie AG, P.B. 3 32, CH-8320 Fehraltorf, Switzerland containing 69.39 % weight/weight (w/w) ethanol, 3.69 % w/w 2-propanol, different amounts of citric acid ranging from 1.0 to 2.5 % and of urea between 0 % and 2.5 % plus polyethylengylcols as skin care compound. The formulations are manufactured following strict quality criteria. Purified water, prepared by a combination of ion exchange and reverse osmosis from municipal water, was used in preparation of all formulations. The microbial count of purified water was under the 100 CFU/ml acceptance criterion specified in European Pharmacopoeia (Ph. Eur.) 8.0. All other components were of Ph. Eur. quality.Tests were carried out in accordance with the DVV/RKI Guideline at 20 °C . One parThe different formulations of the new hand rub based on ethanol, citric acid and urea were screened undiluted (80.0 % due to the addition of test virus suspension and interfering substance) against PV, AdV and polyomavirus SV40 as non-enveloped test viruses of the Guideline of DVV/RKI in the presence of FCS with a fixed exposure time of 60 s. The ethanol and 2-propanol amounts were constant  in these assays while citric acid and urea were used in a dose-dependent manner. The concentration of urea varied between 0 % and 2.5 %, whereas the concentration of citric acid ranged from 1.0 to 2.5 %.−5. One part by volume of water of standardised hardness (instead of test virus suspension) was mixed with one part by volume of interfering substance and eight parts by volume of the disinfectant. Aliquots of 100 μl of each test concentration and each dilution were then inoculated into eight wells of a 96-well microtiter plate containing 100 μl suspension of permissive cells. A control studying the suppression of activity was included. The cell cultures were observed for cytotoxic effects for the same incubation time as afterwards used for the quantitative suspension tests. Virus titers were determined using the methods of Spearman [10TCID50/ml including standard deviation. Titer reduction is presented as the difference between the virus titer after the exposure time with the disinfectant and the control virus titer (water). According to the Guideline of the DVV/RKI, a formulation under test conditions must give at least a 4.0 log10 reduction in infectivity titer of test virus (inactivation ≥ 99.99 %) at the recommended concentration and exposure time to be considered active [For determination of cytotoxicity the formulations were serially diluted 10-fold in MEM up to a dilution of 10Spearman  and KaerSpearman  and exprd active , 34.Tests according to EN 14476 were run in parallel to the Guideline of DVV/RKI with PV, AdV and MNV as test viruses of the EN 14476 and the corresponding permissive cells . The maiThe quantitative carrier test according to the Guideline of DVV was performed with clean conditions . The clevice versa. Steeper slopes indicate greater dose-dependency.To estimate differences in the dose-dependency of the virucidal effect of the tested compounds, we calculated the slope of a linear regression model fitted line for urea titration at each citric acid concentration and 10 reduction (Table 10 steps (data not shown). In conclusion, the screening experiments of a novel ethanol-based formulation supplemented with urea and citric acid showed a strong virucidal activity against the three non-enveloped test viruses of the Guideline of DVV/RKI in the presence of FCS.As shown in Table on Table . In caseon Table . For theon Table . To compon Table . Titration Table . Similaron Table  and c. IConsequently, the formulation with the sufficient virucidal activity containing 69.39 % w/w ethanol, 3.69 % w/w 2-propanol, 2.0 % urea and 2.0 % citric acid was tested against several non-enveloped  and enveloped viruses  in the presence or absence of FCS according to Guideline of DVV/RK or in clean conditions according to EN 14476. The results are presented in Table In general, the non-porous surface test method is designed to evaluate the ability of chemical biocides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterial spores on inanimate surfaces. Here, we evaluated the final formulation as described above to inactivate dried vaccinia virus strain Elstree, AdV and MNV as test viruses of the DVV Guideline  within 1The development of broad antiviral spectrum hand disinfectants with activity against all enveloped and non-enveloped viruses is limited by the small number of permissible active ingredients – broad spectrum hand rubs are generally based on alcohol. With ethanol alone in concentrations above 80 % v/v stable non-enveloped viruses like PV and AdV can be inactivated but not polyomavirus SV40 , 37. In 10 to >4.4 log10 reduction when the pH of the solution is lowered from 7.4 to 3.0 [We now report that urea in combination with citric acid can enhance the virucidal activity of ethanol solution (ca 70 %) and inactivate all reference viruses of the DVV/RKI Guideline within 1 min exposure time. Tests with bacteria and fungi are still under investigation for this antiseptic formulation which requires a broad spectrum as shown earlier for a product based on chlorine and alcohol . The vir4 to 3.0 . Citric 4 to 3.0  and has 4 to 3.0 . Inorgan4 to 3.0 .10 reduction against all three test viruses  on carriers, as defined according to DVV Guideline. The activity of the formulation is sufficient for virucidal activity on carriers according to the current version of prEN 16777. Stainless steel carrier methods have shown a good overall reproducibility between different labs , but theHygienic hand disinfection can only be done with intact skin. For dermal tolerance no data for the product developed are available. In another study with a formulation containing 62 % ethanol and 4 % citric acid 9 % of the panelists were not included due to skin irritation . It can In conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in EN 14476 and DVV/RKI Guideline in quantitative suspension tests and inactivates MNV, AdV and vaccinia virus on stainless disk carriers. The formulation contributes a valuable addition to the hand disinfection portfolio. It is of course not possible to test the activity of a hand disinfectant against each virus. The test viruses as mentioned in European Norms or German Guidelines analogous to bactericide testing are representatives for the whole spectrum of relevant viruses. Therefore, the new formulation will not only inactivate the test viruses from the European Norm or German Guideline but is also covering the whole spectrum of all enveloped and all non-enveloped viruses being directly or indirectly transferred by human hands.


Hepatitis E virus (HEV), an enterically transmitted hepatotropic virus, was thought to be non-enveloped for decades. However, recent studies have revealed that the virus circulating in the patient’s blood is completely cloaked in host membranes and resistant to neutralizing antibodies. The discovery of this novel enveloped form of HEV has raised a series of questions about the fundamental biology of HEV and the way this virus, which has been understudied in the past, interacts with its host. Here, we review recent advances towards understanding this phenomenon and discuss its potential impact on various aspects of the HEV life cycle and immunity. Orthohepevirus genus within the Hepeviridae family. At least four genotypes are responsible for the diseases in humans [Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide . HEV is n humans ,3. Genotn humans . Genotypn humans , where in humans . HEV infn humans ,7. Infecn humans .This small RNA virus has been considered non-enveloped since its discovery 25 years ago ,10. HoweCaliciviridae family. With molecular cloning of the HEV genome in 1990 [HEV was initially isolated from patient stool and visualized by immunoelectron microscopy as non-enveloped particles . The vir in 1990 , it was  in 1990 . Based o in 1990 ,18,19,20The naked HEV virions found in feces have a buoyant density of approximately 1.27 g/mL. However, Takahashi and colleagues found HEV in the culture supernatants exhibited a significantly lesser density (about 1.15 g/mL) . SubsequMoreover, because the host membrane completely masks the viral antigens, antibodies are unable to bind and neutralize the circulating virus particles. Notably, the vast majority of, if not all, virus particles released from cultured cells infected with HEV are enveloped, and cannot be precipitated or neutralized by convalescent human serum, unless the virions are first disrupted with detergent ,24. The The 7.2 kb positive-sensed HEV genome encodes three proteins: ORF1, ORF2, and ORF3. ORF1 is a large nonstructural protein involved in HEV RNA replication. ORF2 is a 72 kD capsid protein required for virus assembly and receptor binding. A third viral protein, ORF3, is a small multifunctional protein essential for virus egress. The C-terminus of ORF3 contains a conserved PSAP  late domain motif, through which ORF3 interacts with the tumor susceptibility gene 101 (Tsg101) protein ,26,27. TAlthough the current evidence suggests that HEV release is dependent on the cellular ESCRT machinery, the origin of the eHEV membrane remains uncertain. One recent study concluded that the membrane is derived from the trans-Golgi network (TGN) because it contains the trans-Golgi network protein 2 (TGOLN2) . Since TGiven the differences between eHEV and HEV, it is likely that the two forms use different mechanisms to enter target cells. For non-enveloped HEV, its initial cell attachment is thought to be mediated through heparan sulfate proteoglycans , which aFollowing cell attachment, both non-enveloped and enveloped HEV virions are internalized through a clathrin- and dynamin-2-dependent pathway ,39. HoweHEV is primarily transmitted enterically. Following ingestion, HEV penetrates the gut through a poorly defined mechanism. Although the human gut stands as a likely first target for HEV, there is no compelling evidence that HEV enters and replicates in human gut cells. Subsequently, HEV enters the bloodstream and is carried to its main target organ, the liver, where the virus multiplies within hepatocytes. Newly synthesized virions are released at both the apical  and the basolateral  surfaces of infected hepatocytes. While some are released from the basolateral membrane (in the form of eHEV) into the sinusoidal blood and re-enter the circulation system, the bulk of the virus is believed to be released from the apical surface into the bile canaliculi, where it enters the biliary tract and is subsequently shed in feces.Only non-enveloped HEV virions are detected in bile and feces, but the form of the HEV virion that is released from the apical surface of the hepatocyte is likely the enveloped HEV. Intriguingly, recent studies using humanized mice have shown that ORF3 is predominantly localized to the bile canaliculi in HEV-infected liver, suggesting that the eHEV particles are released from the apical membrane ,37. ThisIn addition to the fecal-oral route, transfusion creates a potential route of HEV transmission from person to person . InfectiSeveral extrahepatic symptoms have been associated with HEV infection, including neurologic , renal  and rheuThe discovery of the enveloped form of HEV in circulation has fundamentally changed concepts about the mechanism of virus infection and pathogenesis. Given the lack of specific treatment for HEV, a better understanding of the role of envelopment in HEV infection may help identify targets for therapeutic intervention. Future studies may lead to a better understanding of the origin and structural components of the eHEV envelope, and the host factors/pathways involved in the eHEV release and re-entry into cells. With the improvement of cell culture systems and the recently developed humanized chimeric mouse model, it is expected that many details surrounding this novel phenomenon will be revealed in the near future.


In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease.Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5′ and 3′ termini, consistent with the genome organization found in other orthomyxoviruses.  The economic impact of worldwide trade in tilapia is estimated at $7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development. Oreochromis niloticus) is the predominant cultured species worldwide , demonstrated sensitivity to ether and chloroform, and obtained a sequence specific for disease through cDNA library screening that predicted a 420-amino-acid (aa) open reading frame (ORF) with no apparent homology to any nucleic acid or protein sequence in existing databases. The virus was tentatively named tilapia lake virus lake virus (TiLV); however, no taxonomic assignment was feasible at that time . Fergusoin situ hybridization, and infectivity studies indicate that TiLV is a segmented, negative-sense RNA virus. TiLV contains 10 genome segments, each with an open reading frame (ORF). Nine of the segments have no recognizable homology to other known sequences; one segment predicts a protein with weak homology to the PB1 subunit of influenza C virus, an orthomyxovirus. Our findings suggest that TiLV represents a novel orthomyxo-like virus and confirm that it poses a global threat to tilapiine aquaculture , Northern hybridization, mass spectrometry (MS), aculture , 13.https://github.com/nygenome/taxmaps) by mapping against the National Center for Biotechnology Information’s (NCBI) nucleotide database, the NCBI RefSeq database  was used to recover terminal sequences in all 10 clusters. Based on similarity in terminal sequences in the individual clusters, we concluded that the clusters represented 10 viral genomic segments and henceforth will refer to them as segments 1 to 10 (GenBank accession no. KU751814 to KU751823) 1823, we coymerases .The nucleotide sequences of the 5′ and 3′ noncoding regions of all 10 segments were aligned using Geneious software 6.8.1 . ThirteeHigh-throughput sequencing and alignment analyses of a TiLV PCR-positive sample from a fish in Ecuador showed 97.20 to 99.00% nucleotide identity and 98.70 to 100% amino acid identity to the corresponding coding region of nucleotide sequences obtained with samples from Israel .The segmented nature of TiLV and the sizes of specific segments were confirmed in Northern hybridization analyses of extracts from E-11 cells infected with TiLV (brain isolate)  and fromin situ hybridization techniques to both liver and brain samples with TiLV-specific probes. In the brain, hybridization signals for segment 1 genomic RNA  RNA extracts from NNV-infected cells or from NNV virions. No cytopathic effect (CPE) was observed following transfection of deproteinized TiLV RNA extracted from cells or virions  and D, sin situ hybridization to identify and correlate the presence of candidate viral genes and proteins. Results presented here indicate that TiLV is an RNA virus with a genome comprised of 10 unique segments. The largest segment has minimal homology to the PB1 segment of the orthomyxovirus influenza virus C. It also contains the major polymerase motifs , 1312, 1e motifs . We specOrthomyxoviridae. As noted above, the 5′ and 3′ noncoding termini of TiLV include 13 nucleotides that are similar in all segments. This organization resembles that observed for the influenza, Thogoto, and infectious salmon anemia orthomyxoviruses  and depletion of rRNA sequences with RiboZero magnetic kits . High-throughput sequencing was performed in parallel on random-primed cDNA preparations from purified virus and infected brain tissue RNA. Sequencing from purified virus on the Illumina HiSeq 2500 platform (Illumina) resulted in an average of ~200 million reads (100 nucleotides [nt]) per sample. Total RNA was reverse transcribed using SuperScript III  with random hexamers. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment . The resulting double-stranded cDNA mix was sheared to an average fragment size of 200 bp using the manufacturer’s standard settings . Sheared product was purified , and libraries were constructed using KAPA library preparation kits  with 6-nt bar code adapters. The quality and quantity of libraries were checked using Bioanalyzer . Samples were demultiplexed using Illumina-supplied CASAVA software and exported as FastQ files. More than 90% of Illumina reads passed the Q30 filter. Demultiplexed FastQ files were assembled to generate reads, contigs, and clusters. Sequencing on the Ion Torrent PGM platform was performed with Ion PGM Sequencing 200 kits on Ion 318 chips , yielding on average ~2 million reads per sample, with a mean length of 177 nt. For Ion Torrent, cDNA preparations were sheared  for an average fragment size of 200 bp and added to Agencourt AMPure XP beads  for purification, libraries were prepared with KAPA library preparation/Ion Torrent series kits (KAPA), and emulsion PCR was performed with Ion PGM Template OT2 200 kits (Life Technologies). Ion Torrent reads were demultiplexed and exported as FastQ files by the Ion Torrent PGM software. After bar code and adaptor trimming, length filtering, masking of low-complexity regions, and subtraction of ribosomal and host sequences, reads were mapped as described for Illumina data.Unbiased high-throughput sequencing was performed on Illumina HiSeq 2500 and PGM Ion Torrent platforms. For library preparations, total RNA was extracted from purified virus particles and infected fish brain tissue samples with TRI reagent . RNA extractions from TiLV-infected tissues included postextraction DNase I (2 U/µg DNA for 15 min at 37TiLV virions were purified by ultracentrifugation through 25% (wt/vol) sucrose cushions. The samples were trypsinized and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on Q Exactive Plus (Thermo Scientific). Peptides were identified by Discoverer software version 1.4, using the Sequest search engine, the Uniprot database, and the putative TiLV proteins as references. All of these analyses were performed at the Smoler Proteomics Center, Technion, Israel.Fragments representing each segment were amplified by PCR using primers shown in in situ hybridizations were performed with ViewRNA probes  and/or Stellaris fluorescent probes  in tissue sections and cultured infected or noninfected cells. Tissue samples were collected from euthanized infected or uninfected fish and fixed in 10% neutral buffered formalin. Specimens were embedded in paraffin and serially sectioned (5 µm thick), fixed in 10% formaldehyde (Fisher Scientific), deparaffinized, boiled in pretreatment solution (Affymetrix), and digested with proteinase K (Affymetrix). Sections were hybridized for 3 h at 40°C with custom-designed QuantiGene ViewRNA probes (Affymetrix). Bound probes were then amplified per protocol from Affymetrix using PreAmp and Amp molecules. Multiple-label probe oligonucleotides conjugated to alkaline phosphatase (LP-AP type 1) were then added, and Fast Red substrate was used to produce signal . Infected and noninfected cultured cells, grown on 2% gelatin-coated glass coverslips, were fixed with 3.7% formaldehyde. Negative-sense probes, derived from segment 3, labeled with Cy3 or with Quasar 670 fluorophores, and compatible with the Stellaris system, were constructed to detect TiLV mRNA . Cells and culture supernatants were collected upon the appearance of cytopathic effect (CPE) . Deproteinized RNA was prepared from cells using EZ-RNA reagent , according to the manufacturer’s instructions. Supernatants were cleared by centrifugation (200 × g at room temperature for 5 min), and virions were pelleted by ultracentrifugation  through a 25% (wt/vol) sucrose cushion. Each pellet was resuspended in 140 µl phosphate-buffered saline (PBS), and deproteinized RNA was extracted using QIAamp viral RNA isolation kit (Qiagen). E-11 cells (~80% confluence in 6-well plates) were transfected with the 2.5 µg of cellular deproteinized RNA or with the entire deproteinized virion RNA preparation, using the TransIT-mRNA transfection kit .Confluent E-11 cell cultures in 25-cmTiLV sequences are available at GenBank under the following accession numbers: KU751814, KU751815, KU751816, KU751817, KU751818, KU751819, KU751820, KU751821, KU751822, KU751823.Figure S1 Amino acid alignment. TiLV segment 1 putative protein amino acid alignment with influenza C polymerase subunit PB1 shows partial and low homology . Download Figure S1, PDF file, 0.1 MBFigure S2 (A) Brain section from naive Nile tilapia was processed as described in the legend to Figure S2, TIF file, 2.7 MBTable S1 TiLV coding peptides identified by mass spectrometry.Table S1, XLSX file, 0.01 MBTable S2 Primers used to generate probes for Northern hybridization experiments.Table S2, XLSX file, 0.1 MBTable S3 in situ hybridization experiments.Sequences of probes to segments 1, 3, and 5 used for Table S3, XLSX file, 0.01 MB


Pineapple mealybug wilt associated virus -1, -2, and -3 . Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses,  Ampelovirus of the family Closteroviridae [Dysmicoccus [Closteroviridae were once thought to have similar replication and gene expression strategies based on work done with Beet yellow virus (BYV) [Grapevine leafroll-associated virus-3 (GLRaV-3), the type member of the genus Ampelovirus, has unique gene expression strategies [Pineapple mealybug wilt associated viruses (PMWaVs) belong to the genus oviridae . At leasoviridae . All themicoccus ,4. Most us (BYV) , but recrategies . To bettAmpelovirus where it is closely related to GLRaV-1 and GLRaV-3, and possesses the typical genome organization of other Ampeloviruses [Closteroviridae have been shown to encode such suppressor proteins at their 3’-terminus of their genomes, which suggests that certain ORFs in the genomes of PMWaV-1 and PMWaV-2 might encode similar RNA silencing suppressors.In Hawaii, PMWaV-2 has an important role in the etiology of MWP, causing severe wilt symptoms and yield reductions in the presence of mealybug feeding, whereas infection by PMWaV-1 alone, with or without mealybug feeding, may cause yield reductions but does not produce wilt symptoms ,7. Phylooviruses . PMWaV-1oviruses . The genTomato bushy stunt virus [Potato virus Y (PVY) [Beet yellows virus (BYV) [Turnip crinkle virus (TCV), bind sRNAs of different sizes [Cucumber mosaic virus (CMV) interacts with ARGONAUTE1 (AGO1), the core of the RISC [Turnip crinkle virus (TCV) targets DCL4 [Cauliflower mosaic virus (CaMV) p6 protein targets the DRB4 protein [Tomato yellow leaf curl virus [RNA silencing is a powerful defense mechanism that plants employ to combat virus infection . To counnt virus , HC-Pro  Y (PVY) , and p21us (BYV)  specificus (BYV) ,21,22,23nt sizes . In the nt sizes ,25. Plannt sizes . These snt sizes . Plants nt sizes ,29,30. Tnt sizes . There athe RISC ; the p38ets DCL4 ; the Cau protein ; and therl virus ,36 targeTomato chlorosis virus (ToCV) [Wheat streak mosaic (WSMV) [Closteroviridae encode multiple suppressors in their genomes, including Citrus tristeza virus (CTV) and ToCV [Beet yellows virus (BYV) [Grapevine leafroll-associated virus-3 (GLRaV-3) [In addition to the great diversity of their mechanisms of action ,38,39,40s (ToCV) , and thec (WSMV) . Many meand ToCV ,47. The and ToCV . Howeverus (BYV)  and p19.GLRaV-3) . Potato virus X (PVX) in an experimental host.Here, we report that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two of these encoded proteins, p20 and CP, target both local and systemic silencing, whereas proteins p22 and CPd, target only systemic silencing. In the related virus PMWaV-1, we found no proteins that have local suppressor activities and only one encoded protein, p61, which has systemic suppressor activity. All of the identified suppressors of local silencing found in PMWaV-2 have relatively weak suppressor activities, but two of these identified suppressors, p22 and p20, were found to enhance the pathogenicity of Tomato bushy stunt virus (TBSV) p19 (pBIC/p19) under the control of CaMV 35S promoter in binary plasmids [N. benthamiana line 16C. Papaya ring spot virus (PRSV) HCPro (pBIC/HCPro) was a gift from Yang Chen . To screen for potential local or systemic RNA silencing suppressors in PMWaV-1 and PMWaV-2, seven ORFs from the 3’-terminus of PMWaV-2  and four ORFs from the 3’-terminus of PMWaV-1  (Ananus comosus) plants by reverse transcription-PCR (RT-PCR) using specific primers that contained suitable restriction sites . All constructs were confirmed by sequencing (University of Hawaii Biotechnology-Molecular Biology Instrumentation Facility) and ligated into the binary vector pBIC between the 35S promoter and the nopaline synthase (NOS) terminator. Constructs were then electroporated into A. tumefaciens strain C58C1 with a Gene Pulser® II system according to the manufacturer’s protocol . To test for pathogenicity, selected ORFs from PMWaV-2 were inserted into the ClaI/SalI site of the PVX vector pGR107 , dsGFP , and plasmids , were giand p24)  were indon sites , and clor pGR107 , kindly N. benthamiana leaf tissues using TRIzol® reagent as instructed . For Northern blot analysis of GFP and PVX mRNAs, 20 μg of total RNAs were separated on formaldehyde denaturing 1.25% agarose gels. The RNAs in the gel was transferred into Hybond® N+ membranes  by overnight capillary transfer in 5× SSC/10 mM NaOH. For small RNA analysis, 20 μg of total RNAs were separated on 15% denaturing polyacrylamide gels containing 7 M urea in Tris-borate-EDTA (TBE) buffer. Following electrophoresis, gels were stained with ethidium bromide (0.5 μg/mL) to visualize RNAs. The RNAs were transferred to Hybond® N+ membranes (Roche) by electroblotting in 0.5X TBE at 500 mA for 1 h. Membranes with transferred RNAs were UV-cross-linked at 1200 μJ in a UV Stratalinker® 1800 . Membranes were stored at 4 °C until probed using digoxigenin-labeled RNA probes. These probes were prepared from in vitro transcription reactions with 1 μg cloned GFP fragment in pTOPO® or PVX coat protein fragment in pGEM-T®-easy vector (Promega), and linearized using Maxiscript®  according to the manufacturer’s instructions. For GFP siRNA hybridizations, the digoxigenin-labeled in vitro transcription-derived RNAs were partially hydrolyzed in sodium carbonate buffer  at 60 °C for 3 h and then neutralized with 3 M sodium acetate (pH 5.2) before use. The membranes were pre-hybridized in Ultrahybe-oligo® (Ambion) for 1–2 h at 40 °C. All hybridizations were performed at 40 °C overnight. Post hybridization washes were as described previously [Total RNAs were isolated from N. benthamiana plants at 2, 3, 5, 7, 8, and 12 days post infiltration (dpi). As expected, two to three days following infiltration strong GFP fluorescence was observed in all leaves that had been co-infiltrated with Agrobacterium containing the 35S-empty vector and Agrobacterium containing 35S-sGFP   and four ORFs from the 3’-terminus of PMWaV-1   were scr35S-sGFP  Similar V-2 ORFs . By 5 toilencing . The p22ilencing . The strilencing . To confilencing ; the GFPe plants ,51. In ccontrol) . HoweverN. benthamiana 16C plants as a narrow red zone surrounding the infiltrated area, and reflects the silencing of the endogenous GFP transcripts constitutively expressed in N. benthamiana 16C plants. To investigate the effects of the ORFs of PMWaV-1 and -2 on this limited local spread, the formation of this red zone was monitored in N. benthamiana 16C plants after the constructs were co-infiltrated with 35S-sGFP  used as positive and negative controls, were infiltrated into wild-type N. benthamiana leaves. At 3 dpi both PMWaV-2 p20 and CP constructs led to the complete disappearance of GFP fluorescence, while 35S-p19 showed retention of GFP fluorescence at high levels. Northern analysis of GFP mRNA levels correlated well with the expressed fluorescence levels  or with 35S-dsGFP in combination with individual ORFs from PMWaV-1 and PMWaV-2. The 35S-p19 silencing suppressor was used as positive control in these infiltration assays. Plants were visualized under UV light at 18 dpi, and the presence of systemic silencing, indicated by red fluorescence (due to chlorophyll) along the veins, was monitored (p < 0.05). These plants maintained their GFP fluorescence, indicating suppression of silencing, for up to 30 dpi. When PMWaV-2 p20 was co-infiltrated with 35S-sGFP, 52% (33 of 63) of the plants displayed GFP fluorescence at 18 dpi (p < 0.05) that was maintained for up to 30 dpi  ,57. To ionitored . It was o 30 dpi . In conto 30 dpi . The inco 30 dpi  and was Agrobacterium containing constructs to be tested were infiltrated into transgenic N. benthamina 16C plants in upper (1) or lower (2) leaves of the same plant, or in proximal (3) or distal (4) zones of the same leaves as depicted in versus the 35% suppression efficiency of p20 from treatment 2; and all ORFs from treatment 3 versus the 42% suppression efficiency of p19 from treatment 4. The 35% and 42% suppression efficiencies were chosen as base values for this comparison because they were the treatments with the highest non-specific systemic silencing suppression percentage. Chi-square analysis using this approach showed a significant difference in suppression efficiency of several ORFs (p < 0.05). To further test whether any of the PMWaV-1 or -2 ORFs could suppress systemic silencing by directly inhibiting the movement of silencing signals, we performed an assay as described by Guo and Ding , in whicPMWaV-2 CP completely suppressed systemic silencing (30 of 30 plants) and leaves maintained green fluorescence throughout the observation period. Northern blot analysis of GFP mRNA and siRNA levels in newly emerged leaves of plants infiltrated with only 35S-PMWaV-2 CP and 35S-GFP, either in the same leaf or in different leaves, and that also exhibited systemic suppression of silencing . TBSV-p19, which is known to inhibit transitivity, was agroinfiltrated with 35S-dsGF and 35S-sGFP as the positive control in all experiments. As expected, the leaf zones infiltrated with 35S-sGFP, 35S-dsGF, and 35S-p19 showed bright GFP fluorescence. However, leaves infiltrated with either PMWaV-2 p20 or CP together with 35S-sGFP and 35S-dsGFP displayed only the red fluorescence due to chlorophyll at 6 dpi, indicating that RNA silencing was occurring in these leaves -derived vector (pGR107). Although it failed to show local suppressor activity, PMWaV-2 p22 was included in this study because transient agroinfiltration with PMWaV-2 p22 consistently caused necrosis within the infiltrated zone of leaves  were infiltrated with PVX-p20, -CP, or -p22. PVX with no insert, and PVX-p20FS (frameshift mutant of p20) were used as controls. The plants were monitored from 12 dpi for the development of mosaic symptoms characteristic of PVX infection in non-inoculated systemic leaves. At 18 dpi, mild mosaic symptoms developed in many of the plants inoculated with PVX containing various PMWaV-2 ORFs  infected leaves. The incorporation of PMWaV-2-p20 and PMWaV-2-p22 into the PVX genome apparently enabled PVX to accumulate to higher levels in systemic leaves infected with the PVX-PMWaV-2-p20 or PVX-PMWaV-2-p22 constructs .Closteroviridae, and like other members of the Closteroviridae, the viruses associated with MWP have relatively large genomes. Plant viruses of the Closteroviridae have infection cycles that, from the time of initial genome expression to its replication, assembly, and cell-to-cell and systemic movement, are usually of relatively long duration [Among the many plant diseases of economic importance, mealybug wilt of pineapple (MWP) is one of the most complex viral diseases for which the etiology is not well understood . MWP is duration . Becauseduration . One sucduration . Other eduration ,67.closteroviruses, the products of these ORFs may be involved in suppression of RNA silencing. Using an Agrobacterium co-infiltration system in Nicotiana benthamiana, we have analyzed several 3’-ORFs of PMWaV-1 and -2 for their ability to suppress RNA silencing in this model host plant. Both the p20 and CP ORFs of PMWaV-2 were shown to have local and systemic suppression activity, while the p22 and CPd ORFs of PMWaV-2 were shown to suppress only systemic silencing. In contrast, none of the PMWaV-1 ORFs that were tested suppressed local silencing, but the p61 ORF was shown to have systemic suppressor function. We found that both the p20 and CP ORFs of PMWaV-2 were effective against (+) sense sGFP-induced silencing; their suppression effect diminished by 7 to 8 dpi, as evidenced by the disappearance of GFP fluorescence and the concurrent reduction of GFP RNA levels in infiltrated leaves. The PMWaV-2 p20 ORF effectively reduced the accumulation of GFP siRNAs until 5 dpi while the PMWaV-2 CP ORF did not cause such a reduction (Citrus tristeza virus (CTV) [Grapevine leafroll-associated virus-3 (GLRaV-3) [Broad bean wilt virus 2 also do not prevent siRNA accumulation [Tobacco rattle virus (TRV) [Bioinformatic analysis of PMWaV-2 indicated that three ORFs  near the 3’-end of the genome have functions that are not known . It is peduction . Similarus (CTV) , p19.7 sGLRaV-3)  and the mulation . When thus (TRV) . Howeverus (TRV) .The local RNA silencing pathway may be targeted by PMWaV-2 p20 downstream of dsRNA conversion since the PMWaV-2 p20 ORF prevented siRNAs arising from sGFP but not dsGFP. PMWaV-2 p20 also inhibited silencing induced by low levels of hairpin-dsGFP, suggesting that this protein may also act at some point before the formation of dsRNAs. However, the complete mechanism remains to be elucidated. The silencing of (+) sense sGFP-RNA requires the conversion to dsRNA by the action of RDR6 ,70,71, iSuppressors such as p19 of TBSV and p21 of BYV protect target RNAs by reducing siRNA levels, thus preventing the formation of the 21–22nt RNAs that guide the endonucleolytic RNA-induced silencing complex (RISC) to degrade targeted RNAs . The incRY and P1CF proteins of Cocksfoot mottle virus [Tomato yellow leaf curl China virus [All of the tested ORFs caused the development of a distinctive red zone surrounding the area of infiltration that was evident at 8 dpi, suggesting the inability of all these proteins to prevent short distance spread of the silencing signal . The PMWle virus , where ile virus . Howeverna virus . N. benthamiana plants at 8 dpi. Both sizes of siRNA accumulated to approximately the same levels (Rice dwarf phytoreovirus that has the ability to suppress systemic silencing induced by sGFP but not systemic silencing induced by dsGFP [Short (21–22nt) and long (25nt) siRNAs are involved in the cell-to-cell and long distance movement of silencing signals, respectively ,31. We ae levels , suggestby dsGFP .N. benthamiana. Although the precise nature of the systemic silencing signals has not been determined, it is known that the signal/signals move cell-to-cell through plasmodesmata and systemically through phloem [Rice yellow stunt rhabdovirus (RYSV) also suppresses systemic silencing through interference with the secondary amplification of the silencing signal by interaction with RDR6 [Four of the seven ORFs from PMWaV-2 and one ORF from PMWaV-1 were shown to block or interfere with the movement of the systemic silencing signal in h phloem ,84, and,h phloem . The ideith RDR6 . Turnip yellow mosaic virus have been shown to interfere with miRNA-mediated silencing pathways resulting in developmental abnormalities manifested by visible symptoms [Many viral proteins that were originally identified as determinants of pathogenicity or virulence factors have been later identified as suppressors of RNA silencing . The facsymptoms ,41.The comparative and systemic analyses for suppressors of RNA silencing encoded in the genome of PMWaV-2 have led to the identification of two weak suppressors of local silencing, CP and p20, and four proteins CPd, CP, p22, and p20 that have suppressor of systemic silencing function. PMWaV-1 was found to encode only one protein, p61, with systemic silencing suppressor activity, and no proteins with local suppressor activity. The results presented in this study support our hypothesis that the involvement of PMWaV-2, but not PMWaV-1, in the etiology of MWP  might be


The abilities of the virus to cause systemic infection, to transmit to numerous new hosts via droplets or aerosols and to suppress the host immune response for several months or even years after infection make measles a remarkable disease. This review briefly highlights current topics in studies of measles virus host invasion and pathogenesis.Measles virus is a highly contagious negative strand RNA virus that is transmitted via the respiratory route and causes systemic disease in previously unexposed humans and non-human primates. Measles is characterised by fever and skin rash and usually associated with cough, coryza and conjunctivitis. A hallmark of measles is the transient immune suppression, leading to increased susceptibility to opportunistic infections. At the same time, the disease is paradoxically associated with induction of a robust virus-specific immune response, resulting in lifelong immunity to measles. Identification of CD150 and nectin-4 as cellular receptors for measles virus has led to new perspectives on tropism and pathogenesis. In vivo studies in non-human primates have shown that the virus initially infects CD150 Morbillivirus, the subfamily Paramyxovirinae and the family Paramyxoviridae. MV is an enveloped virus with a single strand, non-segmented negative sense RNA genome and exclusively causes disease in old- and new-world non-human primates (NHPs) and humans. Like all morbilliviruses, MV is highly contagious and is transmitted via the respiratory route ,7827,78]+ memory T- and B-cells, resulting in “immune amnesia” [Based on observations in experimentally infected NHPs we proposed an alternative model explaining measles immune suppression, based on the preferential infection and subsequent immune-mediated depletion of CD150amnesia” ,88. The Mechanisms underlying MV entry, dissemination, transmission and immune suppression as discussed in this review are illustrated with images from experimentally infected NHPs in Measles has caused a high number of fatalities throughout history. Recombinant viruses expressing fluorescent reporter proteins have given us the means to study and understand the virus and its pathogenesis from a new perspective. However, these advances not only leave some old mysteries concerning measles pathogenesis unexplained, but also give birth to new questions. Since global eradication of measles is planned for the near future, studies on MV tropism and pathogenesis not only remain important, but also become urgent .


Pestivirus species and were closely related to CSFV. Cross-reactions would interfere with classical swine fever diagnosis in pigs.To determine why serum from small ruminants infected with ruminant pestiviruses reacted positively to classical swine fever virus (CSFV)–specific diagnostic tests, we analyzed 2 pestiviruses from Turkey. They differed genetically and antigenically from known  Flaviviridae that have a highly variable single-stranded positive-sense RNA genome of ≈12.3 kb –1, BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV), as well as a growing number of additional tentative Pestivirus species. CSFV is the causative agent for classical swine fever, which is notifiable to the World Organisation of Animal Health because it is highly contagious and can cause great loss of pigs  and were subjected to commonly used virus neutralization testing (VNT) . Equal oThe complete genome sequence of Aydin/04 was determined as reported previously  and Burdur/05 (KM408491). For further analyses, reference sequences were obtained from GenBank . GeneticSystematic antigenic characterization was performed by using cross-neutralization assays . For thi6 50% tissue culture infectious doses of isolate Aydin/04 and given a booster of 3 × 107 50% tissue culture infectious doses 2 weeks later. Pigs showed no clinical signs of disease, no fever, no platelet or leukocyte depletion, and no viremia (data not shown). For all 3 animals, strong seroconversion was found .Because of their close relationship to CSFV, it was of particular interest to determine the ability of these ruminant pestiviruses to infect pigs and induce clinical disease. Therefore, 3 clinically healthy and pestivirus uninfected weaner (6 weeks of age) piglets were inoculated with 1 × 10Several new genetically diverse groups of pestiviruses have emerged in domestic livestock and wild animals, adding to the continuously growing list of approved and tentative pestivirus species . The genAlthough these novel pestiviruses are the closest known relatives of CSFV, experimental infection of pigs with Aydin/04 did not result in detectable viremia and clinical signs. Nevertheless, these ruminant pestiviruses are candidates for a switch to porcine hosts after ongoing virus evolution, which would have severe consequences for serologic diagnosis of classical swine fever, affecting control and monitoring programs performed in many parts of the world.


Aedes sp. as vectors. The disease has several clinical manifestations, from the mild cold-like illness to the more serious hemorrhagic dengue fever and dengue shock syndrome. Currently, there is no approved drug for the treatment of dengue disease or an effective vaccine to fight the virus. Therefore, the search for antivirals against dengue virus is an active field of research. As new possible receptors and biological pathways of the virus biology are discovered, new strategies are being undertaken to identify possible antiviral molecules. Several groups of researchers have targeted the initial step in the infection as a potential approach to interfere with the virus. The viral entry process is mediated by viral proteins and cellular receptor molecules that end up in the endocytosis of the virion, the fusion of both membranes, and the release of viral RNA in the cytoplasm. This review provides an overview of the targets and progress that has been made in the quest for dengue virus entry inhibitors.Dengue fever, a reemerging disease, is putting nearly 2.5 billion people at risk worldwide. The number of infections and the geographic extension of dengue fever infection have increased in the past decade. The disease is caused by the dengue virus, a flavivirus that uses mosquitos  Aedes mosquitoes, A. aegypti and A. albopictus, which are distributed in tropical and subtropical areas and are widespread in urban and rural areas [ A. albopictus can be found in temperate countries [Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome are caused by the dengue virus. According to the World Health Organization (WHO), dengue is one the most common mosquito borne diseases in the world . It is eal areas . At presountries .There is some uncertainty in the number and distribution of dengue cases due to the lack of reliable information and misdiagnosis and/or misreporting, thus emphasizing the importance of compiling more extensive records on dengue transmission . It is eThe pathogenic flaviviruses primarily include the four dengue serotypes, the Yellow fever virus, the West Nile virus, the Tick borne encephalitis virus, the Murray valley encephalitis virus, and the Japanese encephalitis virus. Dengue virus has an icosahedral symmetry, with diameter between 500 Å (mature virion) and 600 Å (spiky immature virion) . The virThe structural proteins include the capsid (C), premembrane (PrM)/membrane (M), and envelope glycoprotein (E) that contains three main domains. These proteins are involved in the formation of the viral particle. The dengue virus membrane M protein has three portions, an extended N-terminal loop, an amphipatic perimembrane helix, and a pair of transmembrane helices . The capThe entry of the dengue virus into the host cell is a complex process, mediated mainly by E glycoprotein. The first step of dengue virus entry is the binding of the viral E glycoprotein to a cellular receptor and/or attachment factors . SeveralThe dengue virus entry pathway is highly dependent on the cell type and viral strain. Although evidence suggests that the main way of entry for dengue virions is receptor initiated-clathrin mediated endocytosis , direct Two broad directions have been undertaken to fight this disease. Most community level efforts are focused on reducing the mosquito populations, while at the patient level researchers are trying to develop vaccines and antivirals. The fight to reduce mosquito densities has been partially successful in some places, but the rapid development of resistance to chemicals and many other operative hurdles hamper the efficacy of these programs . More soThe ideal solution for fighting dengue virus infection would be by developing a prophylactic vaccine. However, the fact is that current candidates have failed to give protection against all four serotypes, emphasizing the notion that a therapeutic agent—an antiviral drug—may be a key tool to fight the disease. An effective vaccine must protect against all four serotypes; otherwise, vaccinated individuals might be at risk of developing the severe form of dengue due to the phenomenon known as antibody-dependent enhancement (ADE). In this scenario, nonneutralizing antibodies opsonize the virions and mediate augmented entry and infection of cells expressing the Fc gamma receptor , 25. ThiCurrently, there is no approved antiviral drug against the dengue virus. Several research groups have developed different approaches to identify molecules that inhibit dengue virus infection by targeting several structural and nonstructural viral proteins and its cellular receptors. One attractive approach could be blocking the virus before it enters the target cell. In this scenario, nature provides a proof of concept, as dengue virus generally induces the production of highly effective neutralizing antibodies in convalescent patients. The putative antiviral molecules targeting entry would not need to enter the cell, thereby relaxing structural and other constraints in the search and design of these molecules. Blocking the virus before entry could also reduce the hyperactivation of the immune system produced during the triggering of ADE. Dengue virus tropism in humans involves cells from liver and immune and endothelial systems. Other affected organs are lungs and brain (reviewed by ). Permis de novo viral infections [Several approved antiviral drugs show that entry inhibition is a feasible strategy to fight viruses. The anti-HIV drug maraviroc binds the virus coreceptor CCR5 , while efections . This reβ sheets and a short transmembrane domain for insertion into the viral membrane. This protein is highly flexible, undergoing significant conformational changes during the maturation and the fusogenic stages of the viral life cycle [The E glycoprotein is a class II fusion protein, mainly composed of fe cycle , 33, 34.fe cycle .β-strand barrels containing two insertion loops. The dimerization domain, domain II, contains hydrophobic sequences that are conserved among all flaviviruses. These hydrophobic sequences, also known as fusion peptides, are responsible for the insertion of the rearranged E trimer into the cellular membrane during fusion [β barrel configuration, connecting with domain I [Flavivirus virions structural studies show that the E glycoprotein is formed by three domains, in addition to a membrane proximal stem and a transmembrane anchor , 34, 36.g fusion , 36–38. g fusion . The strdomain I .β-strands, while DENV-2 has an extra β-strand [Structural studies using X-ray crystallography and NMR solution structures of the domain III have shown that DENV-3 and DENV-4 contain nine β-strand , 40, 41.β-strand . While tβ-strand , 34. Theβ-strand . Severalβ-strand , 45.α-helices (EH1 and EH2) connected by a conserved sequence [The E protein stem region is located at the C-terminus of the protein, from residues 395 to 450, and it is part of an outer lipid leaflet of the viral membrane . This resequence , 38. Thesequence . The arrsequence . The steβ-strands (β-hairpins) located at the hydrophobic pocket is the key structural element for the initiation of the E glycoprotein rearrangements, induced by a low pH environment in endosomes [The hydrophobic pocket is located in the domain I-domain II interface. This region plays an important role in E glycoprotein rearrangement, acting as a hinge that allows the exposure of the fusion peptide located in the domain II , 36, 46.The E glycoprotein secondary and tertiary structure suffers significant rearrangements in order to adopt the fusogenic conformation . This coDengue virus E glycoprotein adopts two main conformations: dimeric in the mature virion and trimeric in the immature virion and during the adoption of the fusogenic conformation inside the endosome , 34, 36.The conformational changes leading to the formation of E glycoprotein trimer are thought to be initiated when low pH in the endosome is sensed by five histidine residues which are conserved among flaviviruses. These histidines are distributed in the three domains of the protein and in the stem region , 50, 51.Other important structural characteristics of the E glycoprotein of DENV-2, identified by X-ray crystallography and cryo-EM, are the glycosylated Asn153 in domain I and Asn67 in domain II , 36. TheThe most important target molecules on the cellular side of the dengue virus entry process are the attachment factors and receptors . SeveralIt has been suggested that binding of the virus to the cell may require multiple, sequential interaction with several types of receptors. DC-SIGN and glycosaminoglycans (GAGs) are the first line of attachment factors . A seconThe DC-SIGN is a type II transmembrane protein that is abundantly expressed in immature dendritic cells. It is a C-type lectin with an extracellular domain that binds mannose containing carbohydrates with high affinity. DC-SIGN is involved in dendritic cell migration, T-cell priming, and antigen recognition and presentation . DC-SIGNThe interaction DC-SIGN-DENV has been proposed as an attractive target to tackle the infection . A recenHeparan sulfate (HS) belongs to the family of glycosaminoglycans (GAGs), which are very abundant in cell membrane proteoglycans. HS is composed of chains of disaccharides with uronic or L-iduronic acids and an O-sulfated glucosamine derivative . It is wγRs). Evidence suggests that DENV uses two different ways to enter DCs, a primary infection through DC-SIGN into immature DCs and a secondary infection through FcγRs in mature DCs [γRs entry pathway, some authors consider these cellular molecules as receptors for the virus.Besides DC-SIGN, dendritic cells (DCs) also express Fc gamma receptors , although similar peptides from other flaviviruses are not effective against dengue viruses [50: 8 μM).Peptides based on the sequences of the stem region (residues 419 to 447) of all dengue serotypes showed cross-reactive antiviral activity  against dengue virus type 2 in BHK-21 cells [Several tetracycline derivatives have shown affinity for the hydrophobic pocket. These molecules were identified using GEMDOCK, a tool used to predict potential ligands for the active site of a target protein. The compounds doxycycline and rolitetracycline showed a significant21 cells . The bin50: 12 μM, 1.2 μM, and 1.7 μM, resp.). As suggested by docking simulations, the SA-17 molecule appears to bind to the hydrophobic pocket, interacting with amino acid residues crucial for the fusion of the membranes  [The compound SA-17, a derivative of the anthracycline antibiotic doxorubicin, showed antiviral activity against dengue virus serotypes 1, 2, and 3 (EC Gln200) , 90.50: 0.068 to 0.49 μM) and three additional flaviviruses [ in vivo dengue viremia animal model revealed that this compound precipitates in the gastrointestinal tract [Using high throughput docking screening for hydrophobic pocket ligands, a thiophene-pyrimidine molecule (“compound 6”) was also identified as a potential dengue virus entry inhibitor. After optimization, one compound was able to inhibit all dengue serotypes in BHK cells . This molecule showed suppression of the fusion of membranes mediated by E glycoprotein, most likely through interaction with Lys128 and Gln52.Anc pocket . The com in silico docking screening, using the hydrophobic pocket as target [50: 1.2 μM, 3.8 μM, and 1.6 μM, resp.) and very low in vitro cytotoxicity (CC50: >100 μM). This and other studies suggest that compounds with central thiazole rings may be critical to the observed antiviral activity targeting the hydrophobic pocket [β-OG pocket binder. Additional experiments demonstrated that P02 binds E glycoprotein and has antiviral activity (IC50: 13 ± 3 μM) [The compound A5 was also identified by ans target . This moc pocket . Using a ± 3 μM) . in silico identification of potential drugs is the rational design of inhibitor molecules. Structural data from prefusion dengue virus E glycoprotein was used as a model for the design of new DENV entry peptide inhibitors [50: 7 μM and 8 μM, resp.). Both peptides were shown to bind E protein causing structural changes on the surface of DENV-2 virions.A different approach for thehibitors . The reshibitors . Two pep50: 10 μM and 13 μM, resp.), probably due to competition for cellular receptors [50: 35 μM) [E glycoprotein domain III was identified as a putative receptor binding domain that also stabilizes the E glycoprotein structure. Besides being the main target for neutralizing antibodies, several studies have shown that soluble domain III by itself may act as an antiviral through diverse mechanisms. Soluble domain III acts as a dominant negative inhibitor of class II fusion proteins, possibly by interacting with a core trimer intermediate and interfering with the folding back of the lipid bilayers . Purifieeceptors . Further: 35 μM) .50: 4.7 μg/mL) [β-d-(1→3) glucan backbone and piperidine-N-sulfonic acid groups. It was first identified as an inhibitor of HIV-1 entry and propagation [50: 7 μg/mL) [Similarly, cellular receptor molecules that interact with domain III may also have antiviral activity. One such molecule, the sulfated polysaccharide fucoidan, blocks DENV-2 virus entry into target cells by binding to E glycoprotein domain III and competing with the cellular receptors (IC7 μg/mL) . The higpagation . Ichiyam7 μg/mL) . DENV-2 Hippeastrum hybrid), GNA , and UDA (Urtica dioica), showed strong antiviral activity against all serotypes of DENV  [50: 19 μM) [Some studies have shown that carbohydrate binding agents (CBAs) possess antiviral activities against DENV, because its entry into human target cells is carbohydrate-dependent . CBAs al, resp.) . Pradimi: 19 μM) . This mo50: 0.3 μg/mL) [50: 0.12–20 μg/mL) [A recent study has shown that the GAGs heparin and chondroitin sulfate E inhibited DENV in BHK cells, unlike the parent compounds chondroitin sulfate A and C (lowest EC3 μg/mL) . Heparin0 μg/mL) . These cβ-galactopyranose and (1–4)-α-D-galactopyranose. The iota-carrageenan shows an inhibition of DENV-2 multiplication in infected Vero cells (EC50: 0.4 μg/mL) [ Escherichia coli inhibits dengue infection in human dermal microvascular endothelial cells (EC50: 111 nM). This molecule interacts with E protein domain III, competing with heparan sulfate proteoglycans [ in vivo murine model of dengue viremia [ M. scabrella (BRS) and L. leucocephala (LLS) were found to be active against YFV and DENV-1, LLS being about ten times more active in C6/36 [Other heparan sulfate-like molecules also interfere with the entry and multiplication of DENV. Carrageenans are molecules formed by linear chains of alternate (1–3)-4 μg/mL) , 103, proglycans . Lee et  viremia . Two sulin C6/36 .50: 6.9 μM). It was reported that this molecule targets an early stage of infections and was able to reduce antibody-dependent enhancement (ADE) in vitro. In fact, in vitro inhibition of ADE has also been reported for several antiviral compounds [More recently, LCTA-949, an aglycon derivative molecule from the teicoplanin antibiotic, was found to possess antiviral activity against flaviviruses, including DENV-2 . The antompounds , 107.50: 13.1 and 7.8 μM, resp.). It was suggested that these molecules can not only directly inactivate viral particles before attachment but also interfere with the entry process by binding the viral E glycoprotein and blocking interaction with GAGs.In a recent study, Lin et al. tested two hydrolysable tannins, chebulagic acid and punicalagin, as possible dengue virus inhibitors . The two50: 14–47 μM). Notably, although the compound interferes with an early step during entry, it seems also to enhance the binding of the virions to the cell surface.The antiviral activity of sea grass derived natural product, zosteric acid, and several synthetic derivatives was tested by Rees and colleagues . The derThe E glycoprotein is the natural target of the humoral response against the virus. As naturally elicited antibodies do not provide protection across different serotypes, other approaches have been tried such as monoclonal antibodies (mAbs) and engineered derivatives like antibody fragments as another strategy for the blocking of dengue virus entry. It has identified three main epitope regions within dengue virus E glycoprotein: A (located at domain II), B (domain III), and C (domain I). The domains A and B epitopes allowed the generation of monoclonal antibodies with higher biological activity against dengue virus. Antibodies that were reactive to domain A epitopes neutralize the virus and may block virus cell membrane fusion. The domain B epitopes elicited mAbs that were potent neutralizers of virus infectivity and blocked hemagglutination; however, they did not block virus mediated cell membrane fusion .γR [Most anti-DENV mAbs neutralize at least in part, by disrupting the virus ability to bind to mammalian cellular receptors. The mAb 1A1D-2 recognizes a partially hidden epitope  and inhibits dengue virus attachment. The possible inhibition mechanisms could be due to altering the distances between the glycans present in the E glycoprotein and impairing the binding to the DC-SIGN receptor . Domain γR .50: 0.016–2.8 μM) and against several other flaviviruses. The ST-149 binds to the virion capsid protein, being active also in a murine model of dengue viremia.Compound ST-148 was identified by using a high throughput screening for molecules able to block dengue cytopathic effect . NotablyHigh levels of viremia correlate with severe dengue disease ; therefoAn extensive body of experimental evidence indicates that the DENV, while having a relatively simple structural organization, is able to exploit numerous molecules and pathways to infect cells and evade the immune response. Unfortunately, the virus is also able to cause severe, often deadly disease in a fraction of infected individuals. Therefore, the search for antiviral drugs is a top priority, as development of vaccines will also take some time to achieve a potent and balanced response against the four dengue serotypes.Inhibiting the entry of the dengue virus to avoid the infection is an attractive approach to develop potent and specific antivirals. These molecules will exert their effects without having to enter the cells, thereby avoiding strict structural and chemical constraints. This kind of antiviral agents will have the added advantage of potentially limiting the immune system hyperactivation that leads to severe dengue.Dengue viremia is acute during the first days of infection, decaying rapidly after 4-5 days . TherefoIn general, antiviral development is focused on targeting viral components or cellular determinants of the infection. Targeting viral components has yielded the highest number of candidates, yet this strategy is prone to the rapid development of resistance. On the other hand, targeting cellular components reduces the possibility of developing resistance but may have a higher incidence of adverse effects. This approach may take into account cellular factors required for viral replication and maturation or pathogenic processes.The growing body of knowledge about the biology of the virus tends to favor the approach of targeting the viral determinants of the entry process, instead of trying to block all possible cellular receptors and attachment molecules. Particularly promising as a target is the E glycoprotein because of its key role in viral attachment through receptor interaction and subsequent endosomal fusion. Several specific targets within the E glycoprotein are promising, including the stem domain, the hydrophobic pocket, and the domain III. in vivo efficacy and bioavailability are addressed.Another important challenge for the development of antivirals against DENV is the issue of bioavailability of the drugs. Particular drug families may have different chemical and biological properties that may dictate their fate in the organism. However, most of the molecules described here are small compounds in the proof of concept stage of research. Promising compounds may then soon move into preclinical studies where specific questions aboutα-glucosides I and II inhibitor and an iminosugar as possible antiviral treatments [Particularly useful for rapid development of antivirals are the animal models for viral diseases. In the past few years, there have been great efforts in the development of animal models for dengue virus infections. Currently, there are several dengue disease animal models. The AG129 mice deficient in type I and type II interferon receptors have been used to study dengue induced ADE (reviewed in ). This aeatments , 123. Aneatments , 125. A eatments . Howevereatments .


Bos indicus), which were maintained at around the villages of National Research Centre on Mithun (ICAR), Jharnapani, Nagaland, India. A total of 32 Tho Tho bulls were selected and divided into four groups according to their age and each group consisted of 8 bulls, namely, Group I: 18–24 months (n = 8), Group II: 25–36 months (n = 8), Group III: 37–48 months (n = 8), and Group IV: 49 months and above (n = 8). The scrotal circumference and testicular parameters were measured with caliper and tape and age of animals was calculated with dental formula. The body weight of bulls was estimated with Shaeffer's formula. Result revealed that the scrotal circumference was highly correlated with testicular parameters and body weight compared to age. Compared to exotic cattle (Bos taurus), Tho Tho bull's testicular parameters and scrotal circumference were lower. The results of the present study in Tho Tho bulls revealed that scrotal circumference is a useful indicator and is an important selection criterion to determine the testicular development and breeding soundness in young bulls as it is highly correlated with tesicular parameters.The present study was undertaken to assess the relationship between the scrotal circumference and testicular parameters with body weight and age in Tho Tho bulls ( Bos indicus) is a local indigenous bovine species of North Eastern Hilly (NEH) region especially in Nagaland state of India. It is rearing under semi-intensive system. The bull is half of the herd in animal husbandry, which indicates that the sire is one of the parents of all the calves in the herd. Since the bull has more genetic influence (80–90%) of calves it sires, fertile bull selection can be the most powerful method for improvement of the herd. The demand for sperm from outstanding sires has increased with the development of frozen semen technology and the growth of large artificial breeding organizations. Methods to predict sperm production potential and particularly to identify the bulls with high sperm output potential at an early age are important, which is related with testicular parameters and scrotal circumference. Traditionally, Tho Tho bulls have been selected on the basis of growth, rather than on reproductive traits. However, reproduction is one of the most important factors for the economics of livestock production. Generally, testicular measurements at early ages are very useful in selection of breeding sire to collect semen for artificial insemination or breeding purpose. The most important parameters are testicular diameter, testicular length, testicular volume, and scrotal circumference. The males with bigger testes produce more sperm than the males with smaller testes [Tho Tho cattle , Nagaland, India. The study area lies between 25°54′30′′ North latitude and 93°44′15′′ East longitude and at an altitude range of 250–300 mean sea level. The animals were maintained under semi-intensive condition of grazing in the forest during day time and ting them in shed during night time. The experimental animals in shed were fed daily withL × G2/300 × 2.2, where L = body length in inch and G = girth in inch. According to their age, the experimental bulls were equally divided into four groups, each of 8 bulls, namely, Group I: 18–24 months (n = 8), Group II: 25–36 months (n = 8), Group III: 37–48 months (n = 8), and Group IV: 49 months and above (n = 8).Age of the bulls was determined using their dental formula. The body weight of each bull was calculated according to Shaeffer's formula : L × G2πabc, where a = thickness/2, b = width/2, and c = length/2 . Weight of the testes was measured by multiplying volume with 1.038 which is the approximate density of testicular tissue in cattle [Testicular parameters and scrotal circumference were measured with a caliper  and a tape after restraining the bull in the chute . Testicun cattle .P < 0.05 were considered to be statistically significant. Correlation coefficient was estimated among the testicular parameters, scrotal circumference, age, and body weight of the bulls. Differences at P < 0.05 and P < 0.01 were considered to be statistically significant.The results were analysed statistically and expressed as the mean ± S.E.M. Means were analyzed by one way analysis of variance between the different age groups of Tho Tho bulls, followed by Tukey's post hoc test to determine significant differences between the groups using the SPSS/PC computer program . Differences with values of P < 0.05) different between the age groups. It has been reported that the average estimated testicular weight was 18.50, 40.20, and 51.25% greater in bulls aged 25–36, 37–48, and >49 months of age than in 18–24-month-old bulls. Similarly the average estimated testicular volume was 15.50, 28.69, and 33.85% greater in bulls aged 25–36, 37–48, and >49 months of age than in 18–24-month-old bulls. In case of scrotal circumference, the average estimated value was 6.76, 10.58, and 13.35% greater in bulls aged 25–36, 37–48, and >49 months of age than in 18–24-month-old bulls. Correlations between the different measurements for the 32 bulls are shown in P < 0.01) as in exotic cattle [P < 0.05) in Tho Tho cattle bulls. Moreover age . Circumference of the testes was highly correlated with other testicular measurements  higher than Group I (18–24 months) and Group IV higher value than the other three groups. It has been observed in the present study that SC was increased rapidly in young bulls and gradually in mature bulls. But it has been reported that in old age this parameter is decreased due to senile atrophy [In the present study, the mean value of scrotal circumference in Group II (25–36 months) was significantly ( atrophy . In the Scrotal circumference  was incrHigh correlation was observed between testis size and scrotal circumference and based on this collective information, testicular diameter along with scrotal circumference are excellent indicators of spermatogenic function  and this


Tibrovirus currently has two members, Coastal Plains virus and Tibrogargan virus. Here, we report the coding-complete genome sequence of a putative member of this genus, Bivens Arm virus. A genomic comparison reveals Bivens Arm virus to be closely related to, but distinct from, Tibrogargan virus.The new rhabdoviral genus  Rhabdoviridae currently includes 11 genera  and Coastal Plains virus (CPV) members, respectively (Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) in Peachester, Queensland, Australia  cultured at 28°C and 5% CO2 in Eagle’s minimum essential medium  containing 2% fetal bovine serum . Virus RNA isolation and cDNA synthesis from cleared culture supernatants were performed as previously described   and grew a virus stock in escribed . The NexTibrogargan virus.The determined BAV coding-complete genome sequence was found to be ≈93% identical to that of TIBV and contains all of the tibrovirus-typical open reading frames. Based on these data, we confirm that BAV is a tibrovirus that most likely should be assigned to the existing species KP688373.The GenBank accession number of Bivens Arm virus isolate UF 10 is


This study aims to systematically evaluate available evidence regarding direct medical costs of treating cardiovascular (CV) events in Germany after 2003 on an individual patient basis and from a payer perspective. The CV events of interest were myocardial infarction (MI), unstable angina, heart failure (HF), stroke, and peripheral artery disease (PAD).A systematic literature search was performed in the following databases according to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines - Medline, Embase, Centre for Reviews and Dissemination, TIBORDER, and German dissertation database from January 2003 to October 2013. Both observational studies and randomized clinical trials were considered for the review. All values stated in € are inflation adjusted to 2014 € unless stated otherwise.This review included 13 articles. For newly occurred MI patients, the average hospitalization costs during the acute phase were reported to be between € 6790 and € 8918 per admission. In the first year after a MI event, direct medical costs were € 13,838–14,792 per patient. Direct medical costs of chronic HF patients were found to be between € 3417 and 5576 per patient per year. Treatment costs increase with disease progression. The average treatment costs for hospitalized PAD in the acute phase were reported to be € 4963 per admission, € 2535 per patient during month 1–6 after the initial hospitalization, € 1601 in month 7–12, and € 1390 in month 13–18. For stroke of all types, total direct medical costs in the 1st year after an event were reported to be € 13,273 per patient. Total direct medical costs during the 1st year after an ischemic stroke event were € 17,399–21,954 per patient, € 6260 in month 13–18, and € 6496 per year in the subsequent 4 years.MI, unstable angina, HF, stroke and PAD have a high financial impact on the German health care system. Treatment costs of these diseases are mostly incurred during the acute phase of events and tend to decrease over time. Hospitalization and rehabilitation costs were two major cost drivers. Medication costs was one of the smallest cost component reported. Cardiovascular (CV) events are mainly disorders of heart and blood vessels, including coronary heart disease (CHD), cerebrovascular disease (mainly stroke), and peripheral artery disease (PAD). In 2012, CHD is the leading cause of death worldwide, with an estimated 7.4 million people died from it followed by 6.7 million died from stroke. In Germany in 2012, CHD was number one cause with a proportion of 40,2 % on overall death [Acute CHD, stroke, and PAD often require intensive treatments, such as coronary artery bypass grafting (CABG), percutaneous coronary intervention (PCI) and/or fibrinolysis. Often patients experiencing these disorders also need long-term treatment and care, as well as management after the acute stage. All these treatments and procedures lead to a tremendous economic burden to the patients, third party payers, and the society as a whole.In Germany, CV event is highly incident and is one of the most expensive diseases. DEGS1 studies  from 2013 discovered a lifetime prevalence of overall CHD in Germany of 9.3 % among women and men aged 40 to 79 years . 40 % ofHowever, there is a gap in publications with respect to the individual treatment costs of CV events, especially after the implementation of German diagnostic-related groups (G-DRG) in hospitals in 2003 , which gThis systematic literature review aims to provide an overview of the costs of treating individual CV event patients after 2003 from a third-party payer perspective. The CV events of interest were myocardial infarction (MI), unstable angina, heart failure (HF), stroke, and PAD.A systematic literature search was conducted to identify all available publications about individual-level direct medical costs of the CV events of interest in Germany after the G-DRG reform. We adopted the perspective of healthcare providers and German social third party payers, including statutory health insurance (SHI) and statutory nursing insurance (SNI) for cost studies. The scope of the costs was restricted to direct medical costs per affected patient, including hospitalization costs (based on the G-DRG system), outpatient costs (based on the points assigned to the particular treatment published in the German tariff list), medication costs , and rehabilitation and ambulance/paramedic costs (based on the costs reimbursed by the German SHI or SNI).The review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .st 2003 and October 1st 2013 in English or German were searched in the following databases: Medline, Embase, Centre for Reviews and Dissemination (CRD) database, TIBORDER database, and German dissertation database.Publications between January 1Published papers, conference abstracts, and dissertations were all included in this review. Search terms are presented in Table Observational, epidemiological studies and randomized clinical trials (RCTs) with individual cost information were included. For RCT publications, the RCT was included in the review if there was original information about costs. References of the RCT studies were reviewed by hand for sources of original cost data.Exclusion criteria were: setting was not Germany; not SHI perspective or healthcare provider perspective adopted; not including the CV events of interest; costs data before 2003; costs on individual level not available or not calculable; costs reported originated from other studies (in this case the cited study was hand-searched and reviewed); and costs evaluation using health economic models or truncated approaches (no direct data).One researcher conducted the search. If there was doubt regarding the inclusion/exclusion of an article, another independent researcher was consulted.All values stated in € are inflation-adjusted to 2014 € unless stated otherwise.Relevant data was extracted in a Microsoft Excel file. The extracted parameters included study identification, study characteristics , type of CV events, intervention , participants characteristics , direct medical costs , conclusion and limitations of the study.The search resulted in 400 hits. The reviewer removed 50 duplicated papers, leaving 350 papers for title and abstract-based selection. There were 64 articles/abstracts  included for full-text screening, among which 51 articles/abstracts were excluded based on the pre-defined exclusion criteria: 9 studies were excluded because the reported CV event (e.g. atherothrombosis) was not of interest for this study; 3 because German costs were not separately reported; 24 because they failed to report individual costs, instead they reported incremental costs of interventions compared to base treatments; 8 due to lack of individual cost data; 4 studies reported costs based on data before 2003; 3 studies reported costs using a model or using truncated approach based on indirect data. Ultimately, there were 13 studies included in this review, with several studies addressing multiple CV events.The selection procedure was recorded following the PRISMA flowchart Fig. .Fig. 1FlSeven papers and one abstract reported direct medical cost of individual MI patients in Germany –17.nd year onwards).In the literature, methods used to investigate costs of MI include a claims data analysis, a hospital database analysis, medical chart review, and calculations of costs based on healthcare resource utilization from expert panels (or literature review and expert interview) and SHI price. Although there was no clear definition in the included articles regarding acute and long-term treatment of MI, treatment costs can be roughly categorized into costs during acute phase , costs within 1 year after event, and long-term costs after event  patients, the average hospitalization costs during acute phase ranged from € 3622 to 8918/patient , 15–17.p ≤0.0001) [st year after an event. As estimated by Brüggenjürgen and Reinhold, 50–64 % of total costs in the first year were spent on treatment within the acute phase, and 80–85 % of total cost estimates were reached within 6 months after the event [During the first year after an AMI event, direct medical costs of treating AMI patients were reported to be from € 13,838 to 14,792/patient , 17. Totp <0.05), with more than 50 % of costs occurring within 30 days after the implementation [st year costs) [Treatment costs were heavily influenced by treatment strategies and the medical devices used. For example, treatment costs of stent implementation depended on the type of stent used: the use of bare-metal stent (BMS) led to less resource use than drug-eluting stent (DES)  were € 4442, with inpatient cardiac rehabilitation being cheaper than inpatient treatment only . For hosTwo studies reported costs of treating HF from a SHI perspective in Germany , 20. BotDirect medical costs of CHF were calculated to be between € 3417 and 5576/patient per year for patients of all New York Heart Association (NYHA) levels , 20. BieThe review resulted in 5 papers regarding the direct medical costs of treating stroke in Germany , 21–23. For stroke of all types, total direct medical costs in the 1st year after an event were reported to be € 13,273 perpatient . Male past year after the event were € 17,399–21,954 per patient [st year incurred in the acute phase, 66 % were reached within 6 months after the event [st year treatment costs for ischemic stroke patients varied due to the treatment settings where they received rehabilitation treatments: hospitalized patients had around € 3352 more costs per patient per year compared to those treated in rehabilitation centers [st year costs was hospitalization costs , followed by medicines (€ 1915) and outpatient care (€ 1066). For patients who were admitted to a rehabilitation facility for post-stroke rehabilitation, the leading components were hospitalization costs (€ 8844), inpatient rehabilitation care (€ 4788), medicines (€ 1691), and outpatient care (€ 1317). For both groups, hospitalization (including rehabilitation) is the main cost driver for rehabilitation treatment of ischemic stroke patients [For ischemic stroke, total direct medical costs in the 1 patient . 29 % ofpatients .In month 13–18, costs of treating ischemic stroke were € 6260 per patient . During No cost information regarding hemorrhagic stroke and transient ischemic attack was identified.Only one paper was identified reporting direct medical costs of treating PAD .st year costs are consumed in the acute phase and 82.4 % within the first 6 months.Using a Delphi panel to obtain healthcare resource utilization and calculating costs based on reimbursed price, Brüggenjürgen et al. reported that the average treatment costs for hospitalized PAD in the acute phase  were € 4963 per admission, € 2535 per patient during month 1–6 after the initial hospitalization, € 1601 in month 7–12, and € 1390 in month 13–18 , meaningThe key results of all included studies are presented in Table To summarize, MI, unstable angina, HF, stroke and PAD are leading to high direct medical costs in Germany. Treatment costs of MI, PAD and stroke mostly concentrate during the acute phase of the events and tend to decrease over time. Figure This study is, to our knowledge, the first systematic literature review of direct medical costs of treating CV events on individual patient’s level in Germany.As what was found in this review, treatment costs of CV events are high in developed countries. However, the results are not comparable across studies from different countries because of potential various treatment patterns, reimbursement systems, different study years, etc. For example, it was reported that the treatment costs of AMI during 6 months post event were $ 2764–4953  in the US in 2005 . SimilarIn Germany, although treatment costs of CV events are overall high, there was a substantial variance (around € 1000/patient per year) in the costs reported, especially in stroke treatment (up to € 3000/patient per year). Reported costs varied across studies due to the following reasons: 1) different study design, 2) different patient group, and 3) different treatment pattern.Among the 13 studies identified in this review, research methods adopted included claims data analyses, which included relatively large sample sizes from multiple healthcare providers , 22; retAs an example, Brüggenjürgen et al.  reportedIn general, results of RCTs are least representative for the treatment costs of general CV patients in Germany as they normally have strict recruitment procedures based on patients’ baseline characteristics, health problems and medical history . ResultsDue to different study objectives, researchers had variant inclusion and exclusion criteria  for cohorts. For example, Tiemann investigated the costs of primary treatments of AMI patients to compare variations in hospitalization costs over 9 European countries, including Germany . In ordeRestrictions of patient groups were also implemented in several other studies. For example, Reinhold et al. and Kolominsky-Rabas et al. only included patients who experienced their first-ever event  and excluded recurrent events in their studies respectively , 23. HowDifferent mixes of CHF patients were included in the analyses with respect to NYHA level. The studies of Biermann et al.  includedDifferent treatment patterns might influence the cost estimates reported in the studies. To compare the costs and effectiveness of different stents (DES and BMS), Bäumler et al.  only incIn order to compare resource utilization of ischemic stroke patients who underwent rehabilitation in a hospital-based unit or geriatric rehabilitation unit, Abbas et al.  only incTreatment costs are likely to be different across treatment settings . For some diseases, such as AMI which is an acute and life-threatening event and most patients are admitted to a hospital, the results of studies including only hospitalized patients are likely to represent all patient groups. For example, the restriction of the study conducted by Stargardt et al. as inpatient only should not have significant influence on the representativeness of the result of this study . HoweverDespite the general awareness of the economic impact of CV events in Germany, the number of observational studies investigating the per-patient direct medical costs is relatively small. It should be noted that no study was identified for the costs of treating acute HF patients, haemorrhagic stroke, and transient ischemic attack from this literature review. There is a need for more research in this area. This literature review is limited to published studies through October 2013. Future literature reviews should be able to include more recent information on CV costs in Germany.