Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions , similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms. Cryptococcus neoformans (Cn) is known to mainly infect the immunocompromised population and is responsible for the vast majority of cases of cryptococcosis globally[Cryptococcus gattii (Cg), was originally believed to be restricted to healthy individuals in tropical and subtropical countries such as Australia and Papua New Guinea[C. gattii cryptococcosis in temperate regions such as Vancouver Island, British Columbia, and the Pacific Northwest has redrawn public attention to this resurgent fungal pathogen[Cryptococcosis is one of most prominent invasive fungal diseases; it can invade both immunocompromised and immunocompetent hosts and often manifests as life-threatening meningoencephalitis. Among its two major pathogenic agents,  globally. The othew Guinea, 3. The  pathogen, 5.C. neoformans, C. gattii is also an encapsulated budding yeast, but it exhibits distinct morphological, biochemical, and ecological patterns. For example, C. gattii yields both round and bacilliform cells, and it is consistently found inhabiting decaying trees but not bird droppings like C. neoformans[C. gattii is associated with an increased number of cryptococcomas, more neurological complications, and a slower response to therapy, and it usually requires additional diagnostic follow-ups and more frequent neurosurgical intervention, as compared with infection with C. neoformans[C. gattii in terms of its epidemiological and clinical features may be largely due to its distinctive mechanisms of pathogenesis. Previous studies have suggested that C. gattii infection results in defective induction of host immune responses, such as the arrested migration of neutrophils and the reduced expression of several protective pro-inflammatory cytokines[C. gattii also displays some divergent virulence-regulatory mechanisms compared with C. neoformans, such as the antioxidant superoxide dismutase (Sod1) and trehalose-6-phosphate synthase (Tps1 and Tps2)[C. gattii may rely on the variegated expression of virulence genes or some unknown but unique virulence traits to adapt to the host environment in vivo. A complete understanding of its unique mechanisms of pathogenesis is essential for allowing an accurate diagnosis and more appropriate intervention strategies in C. gattii infection, and these mechanisms remain to be further elucidated.As the sibling species of eoformans, 7. Altheoformans. The unicytokines, 10. Furand Tps2)–14. It iet al. first demonstrated that some DUBs might be involved in melanization and pathogenesis in C. neoformans[C. neoformans[C. gattii isolates from the Vancouver Island outbreak, the expression profiles of which display a significant correlation with the cryptococcal intracellular proliferation rate inside macrophage-like cells[C. gattii.Ubiquitination is a critical reversible post-translational modification for regulating cell growth and physiology in eukaryotes. Ubiquiteoformans. Thus, feoformans. Interesike cells. Hence, Cryptococcus gattii using the hypervirulent strain R265 as a model. Deletion of Ubp5 in C. gattii revealed a severe growth defect under both normal and stressful conditions, and it also attenuated virulence in non-vertebrate and mammalian hosts. In contrast to the findings for C. neoformans, enhanced capsule production and melanin synthesis were observed in the C. gattii ubp5Δ mutant, indicating that the utilization of Ubp5 has evolved for distinct regulatory purposes in the virulence composite of these sibling species. Taken together, our study demonstrates the functional convergence and divergence of Ubp5 among pathogenic Cryptococcus species, facilitating a better understanding of C. gattii virulence mechanisms.In the present study, we evaluated the biological functions of Ubp5 in C. gattii gene UBP5 (CNBG_6153) displayed approximately 87% nucleotide identity to UBP5 from C. neoformans var. grubii (CNAG_05650) or C. neoformans var. neoformans (CNBL2960). A phylogenetic analysis of the protein alignment was performed using the deubiquitinase Ubp5 orthologs of the C. neoformans species complex and 10 other fungal species. This protein was classified into distinct clades of basidiomycetous yeasts, ascomycetous yeasts, molds, and zygomycetous molds, consistent with their evolutionary relationship , UCH (amino acids 208 to 525), and USP7 (amino acids 631 to 1103) motifs. These motifs and their arrangement were common in Ubp5 orthologs from all of the analyzed fungi, and the three domains displayed identities of approximately 98%-99% in the C. neoformans species complex, indicating that the protein structure of deubiquitinase Ubp5 was evolutionarily conserved.The tionship . Among tC. gattii, we constructed the Cg-ubp5Δ mutant and its reconstituted strain Cg-ubp5Δ+UBP5 on the background of the R265 hypervirulent isolate. Similarly to C. neoformans[C. gattii, even on rich medium at 30°C. As shown in Cg-ubp5Δ mutant required an incubation of approximately 40 hours to achieve stationary phase, while the WT strain rapidly entered stationary phase by 24 hours. The reconstituted strain of CgUBP5 displayed a partially restored growth rate similar to the WT strain. We also compared the colony sizes of these three strains after a five-day incubation on YPD agar at 30°C. The remarkable differences in colony size further confirmed the decreased growth rate exhibited by the Cg-ubp5Δ mutant . Furthermore, deletion of Ubp5 had a similar effect on the pathogenic factor laccase   . ReconstC. gattii Ubp5 deletion on the total virulence composite, we first performed a survival assay using the murine inhalation model. Immunocompetent BALB/c mice were inoculated intranasally with 105 cells of R265, the Cg-ubp5Δ strain, or the reconstituted strain Cg-ubp5Δ+UBP5. As shown in P = 0.157). In contrast, mice in the Cg-ubp5Δ mutant group did not die even at 80 days after infection, suggesting a significant attenuation of C. gattii virulence due to the deletion of Ubp5 (P<0.001).To gain insight into the overall impact of the C. gattii strain lacking UBP5 resulted in significantly reduced pulmonary fungal burden at different time points after infection (P<0.001), which also displayed a gradual downward trend with an extended duration of infection. Furthermore, no viable colonies were found in the brain of mice infected with the Cg-ubp5Δ strain, in contrast to the other groups. The mice infected with reconstituted strain Cg-ubp5Δ+UBP5 displayed a slight reduction in CFU in both the lung and brain compared with the WT strain but a significant increase in CFU compared with the Cg-ubp5Δ strain  was less virulent than the WT (LT50 = 8 days) or reconstituted (LT50 = 9 days) strains, as determined by survival analysis  and UBC6 (ubiquitin conjugating enzyme), also display significant transcriptional changes under stressful conditions[S. cerevisiae[C. neoformans, many genes encoding ubiquitin-conjugating enzymes are significantly up-regulated in some HOG pathway mutants, while some genes encoding components (such as MAPKKK and Cpb1) of the MAPK and Ca2+/calcineurin signaling pathways also display significant transcriptional changes in the Cn-ubp5Δ mutant[Cryptococcus spp. Finally, the reduced stress tolerance of the Cg-ubp5Δ mutant was also associated with its slower proliferation rate, even in rich media. Yeast cell growth is a complex biological process that relies on the coordination of multiple factors, such as cell division, cell size, nutrients and energy metabolism[S. cerevisiae, deubiquitination is an important modification mechanism that is involved in energy uptake and the cell cycle[C. gattii.The first phenotype observed for the . candida–32. In Conditions, 34. As onditions, 36. Secerevisiae. In C. n5Δ mutant, 33. Theetabolism, 39. In ell cycle, 40. Cg-C. gattii and C. neoformans. For example, the C. gattii ubp5Δ strain was less sensitive to oxidative and NO stress but was more susceptible to cell wall/membrane damaging agents compared with the Cn-ubp5Δ mutant in C. neoformans[Cryptococcus spp. rely on multiple signaling pathways and regulatory mechanisms to respond to each stressor[Cryptococcus spp.However, deletion of the deubiquitinase Ubp5 led to subtle differences in fungal susceptibility to some stressors between eoformans. Cryptoc stressor, and theC. neoformans, CnUbp5 positively regulated both melanin and capsule production, which might be associated with its roles in regulating copper ion metabolism or polysaccharide attachment to the cell wall[C. gattii, which further confirmed the functional reconfiguring of the homologous gene in cryptococcal evolution. Cryptococcus spp. exploit a similar mechanism for both capsule and melanin production, in which vesicles containing the protein components are excreted into the extracellular space and the components are then attached to the cell wall[C. neoformans led to a "leaky melanin" phenotype like Cg-ubp5Δ mutant in this study[Cg-ubp5Δ mutant to Congo Red, we speculate that CgUbp5 might indirectly regulate capsule or melanin production via its role in cell wall synthesis; however, this hypothesis requires further exploration.The functional difference in Ubp5 between the two sibling species was more significant in terms of the expression of other pathogenic factors. In cell wall. Interescell wall–45. Sevecell wall–48. For Cryptococcus neoformans species complex utilizes multiple pathogenic factors to overcome the hostile environment in vivo and cause damage to the host[C. gattii, deletion of deubiquitinase CgUbp5 resulted in significantly attenuated virulence in a mammalian host, although the Cg-ubp5Δ mutant displayed slightly enhanced production of capsule and melanin. Excluding the effect of the reduced thermotolerance, the survival assay in C. elegans model further confirmed the role of CgUbp5 in regulating the virulence composite of C. gattii. The results of our study suggest that the deubiquitinase Ubp5 is essential for the overall virulence phenotype in both C. gattii and C. neoformans but with some common and/or specialized mechanisms[The  the host. Each pa the host. In C. gechanisms.C. gattii lacking deubiquitinase Ubp5 was easily cleared by host pulmonary defense responses. This phenomenon was closely associated with the decreased survival rate of the Cg-ubp5Δ mutant inside macrophages. Alveolar macrophages have been demonstrated to be the first line of host defense, a primary haven for latent infection, and also to function as a Trojan horse for hematogenous dissemination during cryptococcal infection[C. gattii strains recovered from the Vancouver Island outbreak, which are characterized by the upregulation of multiple genes including CgUBP5[C. gattii in the pulmonary space and, thus, its extrapulmonary dissemination.It is believed that cryptococcal spores or desiccated yeast cells are first inhaled into the lungs and then disseminate into extrapulmonary regions in the central nervous system when the host is immunocompromised, 52. In infection. Enhanceng CgUBP5. ConsistC. gattii strain R265. Ubp5 was found to be involved in cellular propagation, the stress response, capsule and melanin production, and thus pathogenicity of C. gattii in both non-vertebrate and vertebrate models. Furthermore, our results revealed the evolutionary convergence and divergence of Ubp5 in these two major cryptococcal pathogens to a certain extent, suggesting that C. gattii exploit some specialized mechanisms to adapt to environments in vivo and in vitro. However, the detailed mechanism by which Ubp5 deletion affects host immunological responses during cryptococcal infection remains unknown. It will be important to explore the potential of deubiquitinase as an anticryptococcal target.In summary, our work supports the importance of deubiquitinase Ubp5 in the virulence composite of C. gattii isolate from the Vancouver Island outbreak[R265, a VGIIa clinical  outbreak, was use outbreak.Cg-UBP5, including the flanking fragments and NEO resistance gene[The primers used in this study are listed in ance gene. The purance gene. Stable ance gene.Cg-UBP5 containing the ORF, promoter and terminator region was amplified from R265 genomic DNA for mutant complementation. The purified PCR fragments were linked to the digested plasmid pCH233 by Xba I using the Infusion®EcoDryTM Cloning System . The linked fragments were then introduced into the mutants by biolistic transformation. Stable colonies were screened on selective medium containing nourseothricin, and finally confirmed by diagnostic PCR and Southern blot analysis.A whole DNA fragment of C. gattii strains, the cells were serially diluted 10-fold (1–106 dilutions). 3 μL suspension of 108 cells/mL were spotted on different stress media, incubated for five days and then photographed. For the high temperature stress test, the yeast cells were incubated at 30°C, 37°C and 39°C on YPD agar. For the oxidative and NO stress test, the cells were incubated on Yeast Nitrogen Base (YNB) agar containing 2 mM H2O2 or 0.75 mM NaNO2 (pH 4.0). To evaluate the response to high salt and osmotic stress, 1.5 M NaCl, 1.5 M KCl or 1.5 M sorbitol was added to the YPD agar. To assess cell wall/membrane integrity stress, 0.5% Congo Red or 0.02% SDS was added to the YPD agar.The yeast cells were cultured to saturation in YPD broth at 30°C, washed twice with 1×PBS buffer, and then quantified using a Countstar Automated Cell Counter. To evaluate the stress response of Cg-ubp5Δ, and Cg-ubp5Δ+UBP5 strains were determined using the Clinical and Laboratory Standards Institute broth microdilution reference method , and Candida parapsilosis ATCC22019 served as a quality control strain.The antifungal susceptibility test was performed as previously reported. The MIC2 at 37°C[7 cells/mL for each strain was spotted on L-DOPA and Caffeic Acid medium[To measure capsule production, fungal cells were incubated on DMEM medium for three days in the presence of 5% CO2 at 37°C. The capid medium and thenC. gattii strains as previously described[6 yeast cells were co-incubated with 105 J744.A1. cells that had been activated with interferon-gamma and lipopolysaccharide in 96-well tissue culture plates. After a 2-hour co-culture, the extracellular yeasts were washed away with PBS buffer, and fresh DMEM medium was added. After 24 hours of incubation, the macrophages were lysed with 0.5% SDS, and viable fungal cells were calculated by quantitative culture on YPD agar at 30°C for 3 days.J744.A1. macrophage cells were used to assay the intracellular survival of different described, 56. In 5 CFU of the mutant (Cg-ubp5Δ), wild-type(WT) (R265), or complemented (Cg-ubp5Δ+UBP5) strain in 50 μL of PBS. All of the mice were monitored daily for signs of infection and sacrificed via carbon dioxide euthanasis based on predetermined endpoints such as weight loss (≥15%), neurological symptoms, and an inability to access food or water. To assess the organ fungal burden, lungs and brains were removed from the sacrificed mice (12 mice per group) after 3, 7, 14, and 21 days.BALB/c mice were intranasally infected according to an established protocol. For theCaenorhabditis elegans (C. elegans) model was also used to evaluate the virulence of each strain under room temperature as previous reported[C. elegans strain N2 Bristol in each group were incubated to the young adult developmental stage on a lawn of Escherichia coli OP50. Subsequently, they were transferred to plates containing WT, mutant, or complemented strains. The viability of C. elegans was determined every day by microscopy.A  reported. BrieflyC. elegans model survival assays were plotted as Kaplan-Meier survival curves and analyzed with the log-rank test using SPSS 18.0 software. The LF50  was also calculated to estimate survival differences in the C. elegans model. The remaining statistical analyses were conducted with the student’s t test or Mann-Whitney test. The results were considered statistically significant when the P value was less than 0.05.The data obtained for the mouse and The animal studies were proved by the Committee on Ethics of Biomedicine Research, Second Military Medical University, and carried out in strict accordance with the recommendations in the Regulations for the Administration of Affairs concerning Experimental Animals of the State Science and Technology Commission (China). Animal model was established under isofluorane anesthesia, and all efforts were made to minimize animal suffering and distress.S1 FigC. gattii R265, KGB80315; C. gattii WM276, XP_003197136; C. neoformans var. grubii (CnVG) H99, AFR99081; C. neoformans var. neoformans (CnVN) JEC21, XP_572460; Ustilago maydis, XP_758786; Candida albicans, KGQ89526; Clavispora lusitaniae, XP_002617519; Candida glabrata, XP_449943; Saccharomyces cerevisiae, EWH16885; Aspergillus fumigatus, XP_748018; Talaromyces marneffei, XP_002147746; Colletotrichum fioriniae, XP_007599895; Fusarium graminearum, XP_009255591; Mucor circinelloides, EPB84371.The alignment of predicted Ubp5 orthologs from various fungal species was performed using the DNASTAR 6.13 ClustalW multiple-sequence alignment. The organism sources and accession numbers (NCBI database) for the protein sequences are as follows: (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


This article contains the data related to the research article “in vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay”   Specifications TableValue of the data•The image data provide various phenotypes of the GFRα1-positive stem/progenitor spermatogonia near GDNF-soaked beads which were transplanted into the interstitium of mouse testes in vivo.•These image data are useful for the estimation of the active state of GFRα1-positive spermatogonia by comparing their phenotypic similarities to those in other testes under various conditions.•These data allow researchers to elucidate the effects of various growth factors in mammalian spermatogenesis in vivo.•This bead transplantation technique will be helpful for the preservation and renewal in sub- and infertile males of various mammal species.1A novel bead transplantation assay into mouse testicular interstitium  [1] prov22.1Wild-type male mice  were used. All animal experiments were performed in strict accordance with the Guidelines for Animal Use and Experimentation at the University of Tokyo .2.2Affi-Gel blue beads  were soaked in a solution of recombinant GDNF  for 1 h at room temperature. To mark the tubular wall adjacent to the transplanted beads, the beads were immersed in DiI  solution for 15 min.For transplantation, the adult testes were gently extracted from the abdominal cavity under anesthesia, and then the GDNF- or BSA (negative control)-soaked beads were transplanted into the testicular interstitium  via vitrified micro-capillary under a dissecting microscope.2.3For whole-mount immunostaining, seminiferous tubule fragments around the transplanted beads were isolated, fixed in 4% PFA-PBS for 8 h at 4 °C, and washed with PBST, as described previously


Poxviridae family. In addition to considering the zoonotic potential of these viruses, we reflect on the broader implications of these findings. Specifically, the potential to explore and exploit this newfound relationship to study coevolution and cross species transmission together with fundamental aspects of poxvirus host tropism as well as bat virology and immunology.Poxviruses are important pathogens of man and numerous domestic and wild animal species. Cross species (including zoonotic) poxvirus infections can have drastic consequences for the recipient host. Bats are a diverse order of mammals known to carry lethal viral zoonoses such as Rabies, Hendra, Nipah, and SARS. Consequent targeted research is revealing bats to be infected with a rich diversity of novel viruses. Poxviruses were recently identified in bats and the settings in which they were found were dramatically different. Here, we review the natural history of poxviruses in bats and highlight the relationship of the viruses to each other and their context in the  Poxviridae. The family is divided into the invertebrate-infecting entomopoxvirinae and chordate-infecting chordopoxvirinae. The latter subfamily is further divided into ten genera and contains many important infectious agents of both animals and humans. The now-eradicated Variola virus  illustrates the potential consequences of poxvirus infections having arguably caused more deaths in human history than any other infectious agent [etc. [Poxviruses are double-stranded DNA viruses with large genomes up to 300 kb) belonging to the family us agent . Aside fnt [etc. ,3 and ma00 kb belnt [etc. . For exant [etc. . Furthernt [etc. . Owing tPoxviruses exhibit a heterogeneous host range with some poxviruses having a very broad host range , and others being very specific . Although some poxvirus genera are known to exhibit broad host tropisms  and are consequently thought to manifest greater zoonotic risks , phylogeBats are an ancient, highly diverse order of mammals that are known to be reservoirs for a large number of viruses . “Bats” Poxviridae, and consider their zoonotic potential. Finally, we speculate on the possible consequences and potential research avenues opened following this marrying of a pathogen of great historical and contemporary importance with an ancient host that has an apparently peculiar relationship with viruses; a fascinating and likely fruitful meeting whose study will be facilitated by recent technological advances and a heightened interest in bat virology.Poxvirus infections have recently been identified in bats, comprising part of the increase in viral families newly identified in this taxonomic order. Here, we review the current evidence of poxvirus infections in bats, present the phylogenetic context of the viruses within the There are three documented detections of poxviruses in bat populations under distinct circumstances (summarized in Eidolon helvum) [E. helvum in Ghana in 2009 contained poxvirus sequences most closely related with Molluscum contagiosum (MOCV) a human-only pathogen  of swabs containing poxviral DNA.Genetic sequence of one bat poxvirus was detected at high prevalence during active surveillance on apparently-healthy African straw-colored fruit bats ( helvum) . Metagenpathogen . Detectexvirus 1 . RetrospNotably, the detection of true poxvirus sequences in this metagenomic study, in which sequences related to multiple genes distributed throughout the genome were found and reconfirmed in individual throat swab samples, is distinct from the detection of poxvirus-like sequences described in other metagenomic studies performed on pooled bat feces, whose presence was ultimately attributed to the presence of other (non-pox) viruses or viral elements integrated into host genomes ,34.Eptescicus fuscus, a microbat) sampled at a wildlife center in the North Western United States [Eptesipox virus was most closely related with Cotia virus, a virus detected in sentinel suckling mice in Sao Paulo, Brazil in 1961  was detected in six adult big brown bats  [Finally, a bat poxvirus was again detected in a clinical setting, in South Australia in 2009. The virus was identified as an incidental infection during investigation of an outbreak of parasitic skin disease in a population of Southern bentwing bats (species) . Bats prspecies) . No furtMolluscum contagiosum virus and Cotia virus respectively  has also been reported in human VARV and Vaccinia virus (VACV) infections [The finding of poxviruses in bats is not unique among wildlife taxa (in fact it would have been more surprising had they not been found to carry poxviruses) and there is no reason to believe they would have greater zoonotic potential than other animal poxviruses. Poxviruses with varying zoonotic potentials have been found in a broad range of wildlife taxa including hundreds of bird species, reptiles, marine mammals, macropods, marsupials, monotremes, ungulates, equids, and primates ,40,41,42kangaroo . Similarir hosts ,45. Notafections . Howeverin vitro host range of isolated viruses such as Eptesipox virus would help inform whether human and further animal cell lines are permissive for infection . Serological and clinical surveillance of human populations for poxvirus infections in geographical regions near detection sites, and/or overlapping with bat home ranges would be a direct approach that would provide samples useful for evaluating multiple candidate zoonoses. Whether bat poxviruses pose a zoonotic threat will likely comprise part of the future research agenda as these investigations are prudent for the discovery of all novel viruses. However, our current knowledge on bat poxviruses does not allow us to make firm predictions about their ability to infect humans.To determine the zoonotic risk posed by bat poxviruses there are, as for other novel viruses, a number of obvious and relatively straightforward investigations that can be done. Full genomic characterization of these viruses to identify known and putative poxvirus host range genes (discussed further below) would be an obvious step. Similarly, testing the in situ), in vitro and in silico studies could elucidate the possible coevolution, cross species infections and mechanisms of host range restriction of bat poxviruses, the implications of which are relevant for bat ecologists, virologists and emerging infectious disease specialists (including those with a specific interest in bats) alike.Irrespective of their potential role as zoonotic agents however, the study of poxviruses in bats opens unique avenues of highly relevant research for multiple research fields beyond the individual host-pathogen relationships. Further field  in the poxvirus phylogeny when compared with other mammalian-infecting poxviruses. It is possible that if evidence of coevolution between bats and poxviruses were present, as has been suggested for the North American poxviruses [It is likely that comparative phylogenetics of bats and poxviruses would inform and deepen our understanding of origins and evolution of both elements. Bats and poxviruses are diverse host and pathogen taxa respectively and given their 0.5 million years of likely co-existence , there ixviruses , this coxviruses ,55,56. IEptesipox virus with its severe disease manifestations, were an emerging cross-species infection it would be useful to identify this rapidly, especially in already endangered species as is the case of the Southern bentwing bat in which a poxvirus was reported. Further to the conservation implications of such research, combining data regarding cross species infection and ecological aspects of host taxa  will likely inform key concepts of virus sharing among bat species, as has been done with lyssaviruses [With concerted research effort to identify reservoir species of bat poxviruses and cross species infections of poxviruses in bats could be identified and would have important implications for both bat and zoonotic-disease specialists. Continued serological and molecular studies of naturally infected bat populations would allow the clinical effect and ecological impact of cross species poxvirus infections in bats to be assessed. We already noted that poxvirus infections across species barriers can devastate wildlife populations , an effect so severe that it was used to control introduced rabbit species in Australia in the 1950s . White naviruses ,61.in vitro refinement of host range definitions beyond detection in the field. Furthermore, full genome sequencing information of poxviruses (now a comparatively easy and cost effective task) would facilitate the in silico identification of poxvirus host range gene orthologues, as recently done by Bratke and colleagues who performed a systematic survey for the presence of known poxviral host range genes on among chordopoxviruses [in vitro tools in place, hypothetical host range genes can be validated, advancing our fundamental knowledge of poxvirus host range restriction.Given the heightened interest in bat virology, further analysis of bat poxviral isolates from both within- and cross-species infections will allow for a deeper understanding of the extent and mechanisms of poxvirus host restriction. Many bat cell lines have now been developed ,64,65,66xviruses . Furtherxviruses ,67. In aFinally, and most speculatively, the identification of genes involved in poxvirus host range restriction in bats may represent a unique opportunity to study bat immunology, which may have broader implications for their confirmed roles as zoonotic reservoirs. Since genes that interplay with the host innate immune system, not those involved with cell entry, are typically responsible for host range determination in poxviruses ,9, the iRecent advances in the study of bats and their viruses as well as the current biotechnological revolution leave us in a position to explore questions of virology as never before. The recent detection of poxviruses in some bat species has occurred consequent to a heightened interest in bats’ role as viral reservoirs. These new findings enable us to ask many exciting and important questions about both bats and poxviruses independently as well as their ecological and evolutionary relationships. Integrating the new and exciting tools of the ‘omics revolution with traditional laboratory and field studies allow us to interrogate these questions as never before.


Sexually transmitted diseases (STDs) data collected in HIV+ patients could be used as indicator of risky sexual behaviour possibly linked to HIV transmission. We described the STDs incidence over time and identified higher incidence factors.All patients in the ICONA Foundation Study enrolled after 1998 were included. STDs considered: any-stage syphilis, human papilloma virus (HPV) diseases, gonococcal and non-gonococcal urethritis, herpes simplex virus (HSV) genital ulcers, vaginitis and acute hepatitis B virus (HBV), hepatitis A virus (HAV), and hepatitis C virus (HCV) infections (only for non-IVDU (intravenous drug user) patients). STDs incidence rate (IR): number of STDs divided by person years of follow-up (PYFU). Calendar periods: 1998–2002, 2003–2007 and 2008–2012. Predictors of STDs occurrence were identified using Poisson regression and sandwich estimates for the standard errors were used for multiple STD events.Data of 9,168 patients were analyzed (median age 37.3 (SD=9.3), 74% male, 30% MSM). Over 46,736 PYFU, 996 episodes of STDs were observed . Median (IQR) CD4/mmc and HIV-RNA/mL at STD: 433 (251–600) and 10,900 . Highest crude IRs were observed for any-stage syphilis , HPV diseases  and acute hepatitis . At multivariable analysis  seems to reduce the risk of acquiring STDs independently of its viro-immunological effect.


RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. Vaccines remain one of the greatest accomplishments of human ingenuity, scientific endeavor, and the combined global efforts of the public health community. The rates of incidence and mortality associated with infection by RNA viruses such as polio, measles, mumps and rubella have declined by greater than 95% compared to pre-vaccination rates . Though Since the development of effective live-attenuated vaccines, new vaccine preparations utilizing well-established vectors, expression of specific viral proteins or components (subunit vaccines), and development of virus-like particles (VLPs), have continued to shape the domain of vaccine discovery and development. The challenge of establishing a safe, immunogenic platform, which induces lasting immunity in the context of a wide variety of viral systems, has resulted in remarkable creativity and variability in the approaches employed. Use of replicating viruses in vaccines, such as live-attenuated or chimeric vector-based platforms, have the benefits of high immunogenicity, lower costs, and ease to transport and administer. Yet, these viruses have the potential to revert to more pathogenic phenotypes and may be under-attenuated in immunocompromised hosts. Conversely, component, subunit, or killed pathogen vaccines have the benefits of generally being safer and can be used to display the most immunogenic antigens. However, the costs, time of development, and weaker induced immune responses present their own challenges to design and implementation. In this review, we describe RNA virus reverse genetics systems and provide an overview of current efforts to use reverse genetics technology in the development of safe and effective vaccines.E. coli and permit the insertion of large DNA fragments to be transcribed under the control of a transcriptional promoter such as T7. BAC constructs have been employed for many years in the recovery of large DNA viruses. However, the genetic stability of these constructs has led to their use in recombinant RNA virus platforms. Several positive-strand RNA viruses and one negative-strand RNA virus have used BACs as a platform for reverse genetic design . In addition, nearly all RNA virus replication strategies are independent from the host cell nucleus and instead reside in the cytoplasm. For positive-strand RNA viruses, such as poliovirus, immediately after entry and uncoating, the genomic RNA is directly translated by host ribosomes to generate viral protein products. Since the virus rarely needs to package additional non‑structural proteins in the virion, most positive-sense RNA virus reverse genetics systems largely focus on delivery of either transcribed genomic RNA into the cell cytoplasm or delivery of cDNA under the control of a viral transcription promoter such as T7 or CMV  3,4,5,6,9,10. Hoc design . Since mPositive-strand RNA viruses have genomes which are infectious upon entry into host cells. Upon entry, host ribosomes translate the viral RNA into one or more polyproteins, which require either host or viral proteases for processing. Although many different reverse genetics platforms have been used to generate positive-strand RNA virus clones, nearly all share the common goal to introduce either sense genomic RNA transcripts directly or cDNA to be transcribed by an included RdRp . Below wPicornaviruses are a family of non-enveloped, positive-strand RNA viruses, which collectively infect a wide range of human and animal hosts. Human picornaviral pathogens cause illnesses, which vary from the common cold to poliomyelitis. The picornavirus virion consists of an icosahedral capsid structure surrounding an RNA genome ranging from approximately 7 to 9 kb in size. Poliovirus and rhinovirus remain two of the most extensively studied picornaviruses to date and have become model systems for the study of RNA virus biology, pathogenesis, and epidemiology. During the 1950’s, extensive efforts began to develop and implement a polio vaccine in the hopes to eradicate the virus since no non-human primate reservoirs are known to exist in nature. The effectiveness of the inactivated  vaccine and live-attenuated (Sabin) vaccines led to increased efforts to develop and design vaccines for many other human pathogens. International vaccination efforts are aimed at eradicating the disease. The success and employed strategies for implementation of vaccines for poliovirus opened the door to evaluating new approaches and efforts to study and understand RNA virus biology. A major step occurred in 1981 when Racaniello and Baltimore introduced a full-length cDNA clone of poliovirus into a cell and recovered infectious virus . AlthougH1 promoting adjuvant induced a cross‑serotype immune response in mice [No commercially available rhinovirus vaccines exist today despite effective reverse genetics platforms for several serotypes. Rhinoviruses remain the primary cause of the common cold worldwide and have been extensively studied for over 50 years. Yet, the great puzzle of developing a broadly protective rhinovirus vaccine involves solving the overwhelming task of developing an immunogenic platform which can provide protective immunity to the greater than 100 serotypes of rhinovirus that currently circulate in nature ,33. The  in mice . The dis in mice ,38,39.Coronaviruses are enveloped, positive-strand RNA viruses which encode the largest known RNA virus genomes varying in size from 26 to 32 kb . Coronavin vitro transcription and capping of a full-length cDNA clone of the coronavirus genome followed by introduction into competent cells by either transfection or electroporation. The coronavirus genome, which is approximately 30 kb in size, is often maintained as fragments in low-copy plasmids. During virus assembly, the fragments are restriction digested and ligated together prior to transcription. This approach has been successfully used to recover clones of many coronaviruses including transmissible gastroenteritis virus (TGEV) [There are currently two common platforms that are used for generating full-length infectious coronavirus clones. The first platform involves s (TGEV) , murine s (TGEV) , NL63 [4s (TGEV) , SARS cos (TGEV) , SARS-lis (TGEV) , and MERs (TGEV) . Anothers (TGEV) . Both ofs (TGEV) .Efforts to design a coronavirus vaccine have focused on a variety of approaches including development of inactivated virus, live-attenuated virus, and a variety of subunit vaccines. One major focus of current coronavirus vaccine efforts is focusing on ways to create a live-attenuated vaccine strain, which combines existing treatments with a less virulent and more stable virus platform. During the SARS epidemic, patients showed little improvement when treated with ribavirin. Recent studies have shown that coronaviruses are resistant to ribavirin treatment due to the presence of a viral exonuclease (nsp14) with proofreading activity ,45. AddiFlaviviruses are small, enveloped, positive-strand RNA viruses that infect a wide range of hosts. Flavivirus genomes are markedly smaller than coronaviruses at approximately 10–12 kb in size, and the transmission of most flaviviruses is dependent upon an arthropod vector . Flavivirus diseases range from asymptomatic to severe neurological disease such as encephalitis, meningitis, and myelitis . Yellow in vitro transcription of full-length or ligated cDNA fragments of the genome or use a bacterial artificial chromosome (BAC) platform . The antigenomes of negative-strand RNA viruses are introduced either as a linearized cDNA or as a plasmid under a T7 promoter and are normally co-transfected with one or more plasmids encoding the nucleocapsid and replicase machinery. Due to these biological requirements to initiate infection, development of negative-strand RNA virus reverse genetics systems has been slower than positive‑strand virus systems. Initial recovery efforts to generate infectious clones involved recovery of viruses using helper viruses, which could supply the viral genes and proteins necessary for replication. However, these approaches made it difficult to isolate the mutants of interest. The first recovery of a negative‑strand RNA virus completely from cDNA was achieved in 1994 for rabies virus ,88. DespParamyxoviruses are enveloped, negative-sense, single-strand RNA viruses which are responsible for a variety of human and animal diseases. Human paramyxoviruses have been identified which are responsible for diseases including measles, mumps, pneumonia, and the common cold. Paramyxoviruses carry a single copy of their genome, which is typically 15 to 19 kb in length. Like other negative-strand RNA viruses, paramyxoviruses must incorporate their replication machinery, including the RDRP, into the virion during assembly. Paramyxovirus reverse genetic systems employ very similar mechanisms. First, the full-length genome or antigenome and helper plasmids expressing nucleocapsid and polymerase proteins are cloned as cDNA and are under transcriptional control by a promoter such as T7 RNA polymerase. The plasmids are co-transfected into permissive cell lines. Earlier reverse genetics systems utilized a co-infection approach with a vaccinia virus expressing T7, however most modern reverse genetics systems use a T7 cell line or transfect a T7 plasmid. Recently, the first BAC-based reverse genetics system for a negative-strand RNA virus was developed for respiratory syncytial virus (RSV) . ReverseThe first paramyxovirus vaccine was developed during the 1950’s. John Enders was able to develop a cultivation system for measles virus and cultured an attenuated measles virus called the Edmonston strain (named after the child from which it was isolated). The Edmonston strain, though initially under‑attenuated, was later adapted and led to the successfully license of a measles vaccine in 1963 . MauriceRespiratory syncytial virus (RSV) was first isolated in 1955 from a chimpanzee displaying upper respiratory illness . Since iOrthomyxoviruses are enveloped, negative-sense RNA viruses whose genomes consist of multiple linear segments and are approximately 12 to 15 kb in size. Similar to paramyxoviruses, many orthomyxoviruses cause respiratory illnesses in humans, most notable is influenza (flu). A key hallmark of orthoymyxovirus evolution is the reassortment of the virus genomes through co-infection of a cell. In order to be transcriptionally active, influenza viruses require a functionally active viral ribonucleoprotein complex (RNP) which consists of the viral genomic RNA, nucleoprotein (NP), and the viral RdRp  . The firS. frugiperda insect (Sf9) cells [For many years, the trivalent vaccine that is currently provided consisted of three separated strains (2 A strains and 1 B strain), which were selected based on the WHO recommendations prior to the next flu season. However, these vaccines were made using either live-attenuated (through cold-adaptation) or inactivated virus grown in fertilized chicken eggs. Next generation vaccination strategies include tetravalent or quadrivalent vaccines, which may be grown in animal cell cultures (rather than chicken eggs) or virus-like particles (VLPs) in cultures of 9) cells ,128,129.9) cells . These r9) cells . et al. have developed an improved platform approach and demonstrated that with current reverse genetics technology, it is feasible to generate a recombinant influenza virus from new HA and NA sequences within 5 days [A major frontier of synthetic biology is the development of new vaccines and therapeutics and the improvement of the implementation and efficacy of those already on the market. One major complication to the successful implementation of vaccines currently is the limitations in time between vaccine design and production. Influenza viruses require seasonal vaccinations and determination of the proper formulations leaves little time for development and implementation. New advances in reverse genetics technology are reducing the potential time of recovery and production from months to weeks. For instance, Dormitzer n 5 days . As new One key limitation to the development of effective live-attenuated vaccines is genetic stability. RNA viruses generally exhibit high mutation rates due to decreased fidelity of the RDRP. Yet, recent studies have shown that codon-usage bias can be used to alter the translation and consequently, replication of viruses. Several groups have shown that substituting non-preferred codons based on host cell codon usage bias, a process referred to as codon-deoptimization, into the genome of poliovirus resulted in reduction of plaque areas and virus yields ,134. By Despite the presence of reverse genetics systems for many human pathogens, not all have resulted in successful vaccine platforms. Advances in biotechnology and key discoveries have led to novel reverse genetic approaches, which may be employed in new generation vaccines. Structural vaccinology or structure-based antigen design has become a common practice for optimizing antigens for display in vaccines . As mentAdvances in biotechnology have shaped the field of virology as much as any other field of science. The capacity to engineer reverse genetics platforms for the study and manipulation of RNA virus genomes has revolutionized the field of vaccine design. We have described here RNA virus reverse genetic systems and past and current efforts to develop vaccines to provide immunity to several human RNA virus pathogens. The incorporation of new advances in reverse genetics technology, adjuvants, non-human models of infection, and surveillance will continue to drive the development of next generation vaccines to pathogens for which we already have vaccination strategies as well as those that we currently do not. Ongoing changes in human demographics and accessibility to health care are likely to modify cost-benefit analyses and provoke allocation of new resources for scientific study and development of vaccines. Despite the ongoing evolution of vaccine design and implementation, the hallmarks of an effective viral vaccine will remain the same: high efficacy, safety, and stability.


Viral diseases are the second most significant biotic stress for sweet potato, with yield losses reaching 20% to 40%. Over 30 viruses have been reported to infect sweet potato around the world, and 11 of these have been detected in China. Most of these viruses were detected by traditional detection approaches that show disadvantages in detection throughput. Next-generation sequencing technology provides a novel, high sensitive method for virus detection and diagnosis.We report the polyadenylated RNA virome of three sweet potato cultivars using a high throughput RNA sequencing approach. Transcripts of 15 different viruses were detected, 11 of which were detected in cultivar Xushu18, whilst 11 and 4 viruses were detected in Guangshu 87 and Jingshu 6, respectively. Four were detected in sweet potato for the first time, and 4 were found for the first time in China. The most prevalent virus was SPFMV, which constituted 88% of the total viral sequence reads. Virus transcripts with extremely low expression levels were also detected, such as transcripts of SPLCV, CMV and CymMV. Digital gene expression (DGE) and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that the highest viral transcript expression levels were found in fibrous and tuberous roots, which suggest that these tissues should be optimum samples for virus detection.A total of 15 viruses were presumed to present in three sweet potato cultivars growing in China. This is the first insight into the sweet potato polyadenylated RNA virome. These results can serve as a basis for further work to investigate whether some of the 'new' viruses infecting sweet potato are pathogenic. Ipomoea batatas L. (Lam.)] originated in South America and was transported across the pacific by Polynesians The sweet potato  Viral diseases are the second most significant biotic stress for sweet potato after the sweet potato weevil Up until 1995, most of the work on sweet potato virus focused on SPFMV, but in the past 18 years, due to the advent of molecular biology, various comprehensive studies on virus composition and the effects of viral diseases were reported I. batatas cv. Xushu 18) were planted in an experimental field at Sichuan University under natural conditions. All of the following samples were collected from symptomless plants: Fibrous roots (FR) at one month after planting; young leaves (YL), mature leaves (ML), stems and initial tuberous roots (ITR) at 1.5 months; expanding tuberous roots at 3 months; harvested tuberous roots at 5 months; newly opened flowers were collected from symptomless drought-treated plants at 4 months.Sweet potato plants (http://www.genomics.cn) for mRNA purification and RNA sequencing (RNA-Seq) with Illumina Hiseq 2000.Total RNA was extracted using the TRIzol Reagent (Invitrogen), and genomic DNA removed with DNase I  according to the manufacturer’s instructions. Then the purity, concentration and RNA integrity number (RIN) of total RNA were measured with a SMA3000 and/or Agilent 2100 Bioanalyzer. The assessed total RNA was submitted to the Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen, China (http://www.ncbi.nlm.nih.gov/Traces/sra)  profiling results ces/sra)  were re-Equal RNA extracted from FR, YL, ML, ITR and stems were reversely transcribed with Moloney murine leukemia virus (MMLV) reverse transcriptase  using Oligo(dT) as primer. The resulting cDNA was subjected to viral sequence amplification and viral gene expression level analysis.Fourteen pairs of primers were designed according to the assembled viral transcripts  using Prdenovo assembly and sequence annotation information were deposited at the Center for Functional Genomics and Bioinformatics of Sichuan University (http://cfgbi.scu.edu.cn/index.html). All of the results described above have been published in 2012 Badnavirus genus: SPBV-A (sweet potato badnavirus A) and SPBV-B (sweet potato badnavirus B) which were suggested to be Sweet potato pakakuy virus (SPPV) . The others were all RNA viruses, in which SPFMV, SPVG, SwPLV, SPV2 and SPVB2 (sweet potato virus B2), YMV (Yam mosaic virus), TuMV-like (Turnip mosaic virus) and SuMV (Sunflower mosaic virus) are from the Potyvirus genus; and NCMV-like  and MMMV (Mikania micrantha mosaic virus) are from Cytorhabdovirus and Fabavirus, respectively. MMMV is also known as Mikania micrantha wilt virus (MMWV) as it was first discovered in Mikania micranthaSeven vegetative tissues were collected from sweet potato cv. Xushu 18 and equal RNA of each tissue sample was pooled together for RNA-Sequencing. A total of 48,716,884 PE reads were generated by Illumina/Solexa Genome Analyzer II. The Sweet potato virus C (SPVC) . Furthermore, at least two distinct transcripts related to SPVG strains were identified , which is the motif of DGE tags. These transcripts belonged to SPVC (5 transcripts), SPFMV (5 transcripts), SPVG (3 transcripts), SPV2 (2 transcripts), SwPLV (1 transcript) and NCMV-like (1 transcript). DGE quantification results showed that different viral transcripts had different expression levels Click here for additional data file.


CASP1 variants suffer from systemic sterile inflammation despite altered enzymatic activity of procaspase-1 and reduced IL-1β release. Most recent findings from our group indicate that the proinflammatory effects of CASP1 variants with reduced or abrogated enzymatic activity could be due to receptor interacting protein kinase 2 (RIP2) mediated increase of NF-kB activation. These findings are additionally supported by a trend to elevated IL-6 and TNF-α expression in patients with CASP1 variants.Patients with unexplained recurrent febrile episodes and CASP1-L265S variant influences proinflammatory cell death (pyroptosis) and IL-1β secretion.The objective of this project is the identification of possible subcellular mechanisms how the We established an in vitro model of a virally transduced human monocytic cell line (THP-1 with shRNA knock-down of endogenous procaspase-1), expressing either wild type or enzymatically inactive (L265S) procaspase-1 fusion-reporter proteins and characterized them after NLRP3-inflammasome stimulation. Using confocal microscopy and in vivo live cell imaging we analyzed the subcellular distribution of fluorescently labeled procaspase-1 wildtype and variant as well as the interaction with ASC.CASP1-L265S variant compared to procaspase-1 wildtype. Furthermore, CASP1-L265S variant revealed a strongly decreased pyroptosome formation and a less intense interaction with ASC (apoptosis-associated speck-like protein containing a CARD) after NLRP3-stimulation. Variant procaspase-1 L265S and ASC formed smaller pyroptosomes than wildtype procaspase-1and ASC.First results revealed a disturbed nuclear localization of the Those findings suggest a model, in which variant procaspase-1 L265S impairs nuclear localization, pyroptosome formation and ASC-interaction, leading all together to reduced IL-1β production and secretion.


KMT2B locus is present in three liver tumors, but absent in their matched adjacent normal samples, indicating that viral integration induced host driver genetic alterations are required on top of viral oncogene expression for initiation and progression of liver hepatocellular carcinoma. Notably, viral integrations were found in many genes, including novel recurrent HPV integrations at PTPN13 in cervical cancer. Finally, we observed a set of HHV4 and HBV variants strongly associated with ethnic groups, likely due to viral sequence evolution under environmental influences. These findings provide important new insights into viral roles of tumor initiation and progression and potential new therapeutic targets.We applied a newly developed bioinformatics system called VirusScan to investigate the viral basis of 6,813 human tumors and 559 adjacent normal samples across 23 cancer types and identified 505 virus positive samples with distinctive, organ system- and cancer type-specific distributions. We found that herpes viruses  that are highly prevalent across cancers of the digestive tract showed significantly higher abundances in tumor versus adjacent normal samples, supporting their association with these cancers. We also found three HPV16-positive samples in brain lower grade glioma (LGG). Further, recurrent HBV integration at the  Much of the community’s collective sequencing capacity has been devoted to cancer genomics over the last several years, with the result being that DNA and RNA data are available for thousands of tumor samples across many different cancer types. The Cancer Genome Atlas (TCGA) Pan-Cancer project has discovered numerous somatic mutations in key cancer genes1245674It is estimated that viral infection contributes to 10–15% of human cancer cases111214151621The Pan-Cancer TCGA data collection is a useful, large-sample resource for studying virus-host interactions and their implications for human cancer. Early results have been obtained232323We obtained 7,372 TCGA RNA-Seq data sets from CGHub, comprising 6,813 tumors and 559 adjacent normal samples across 23 cancer types, the full names and abbreviations are listed in Materials and Methods. We developed a bioinformatics system called VirusScan Methods,  for accu4 and two STAD samples and one COAD sample are positive for both HHV4 and HHV5. Conversely, HBV and HCV are prevalent in LIHC  (We quantified virus abundance by numbers of virus-supporting reads per hundred million reads processed (RPHM). A histogram of HPV read counts shows clear bimodal distribution , suggestctively) .We found that 14.6% of HNSC samples are HPV-positive, with the majority subtype being HPV16. The latter figure is comparable to previous observations2527A virus signature is often observed in the form of a single dominant virus subtype in each individual tumor . This ma4 but complete viral absence in the adjacent normal sample and the other with adjacent normal RPHM > 104 and very low viral presence in the tumor. The clinical-pathological information shows that patient DD-A1EH with only HBV in adjacent normal has a family history of cancer, and sample DD-A11A with HBV in tumor has no family history of cancer, which may indicate different cancer etiologies, i.e., inherited mutations and HBV infection, respectively. We also looked the expression data of 624 cancer genes for the two tumors26Among the 559 adjacent normal samples with available RNA-Seq data, we found 81 tumor-adjacent normal pairs across 15 cancer types that showed varied virus signatures for HBV, HPV and HHV. Chronic infection with HBV or HCV is a known risk factor for liver cancer30We compared gene expression profiles for the tumor-adjacent normal pairs, where both tumor and matched adjacent normal were found to be HBV-positive (RPHM ≥ 100). This criterion returns 6 tumor-adjacent normal pairs .KMT2B (MLL4), suggesting the integration sites in this gene are important for the development of liver cancer, consistent with other studies16Overall, the HBV X protein is the highest expressed gene in both tumors and adjacent normal pairs , followe37EBER-1 gene and a second region that includes genes LF1, BIF1, BALF3, BALF4, and BALF5. EBER-1 has been shown to bind the dsRNA-activated inhibitor of the DNA-dependent activator of IFN-regulatory factors (DAI)EBER-1 and tumor morphology and primary siteEBER-1 is highly expressed in BR-8676. We also detected high expression of LF1, BIF1, BALF5, BALF4 and BALF3 in samples L5-A34H and LN-A49S.We found that a total of 43 samples, including five ESCA, two HNSC, 27 STAD, four COAD, two READ samples, one LIHC, one LUSC and one SKCM samples, are HHV4-positive (RPHM ≥ 100) . Figure RNA2.7, a 2.7-kb RNA gene that inhibits apoptosisRNA2.7 can be regarded as a viral oncogene. It was also expressed in other HHV5-positive samples , four bladder urothelial carcinoma (BLCA), one uterine carcinosarcoma (UCS), five HNSC and one lung squamous cell carcinoma (LUSC) samples , consist samples . For HHV samples .E6 and E7 were expressed in most samples, while E2, E4, and E5 were expressed in fewer samples, and L1 and L2 were expressed only occasionally. HPV16 and HPV18 share similar expression profiles. For three HPV16-positive LGG samples, E6 and E7 were expressed, but E5, L1 and L2 were not , 2) RAD51B (eight samples), 3) PGAP3, ERBB2, and IKZF3 (six samples), and 4) LINC00393 (five samples). We show the location of these common integration sites on MYC region in chromosome 8q24.21, which is a well-known HPV integration site23CASC8 and PVT1 are two genes nearest to MYC with recurrent HPV integration sites. We compared the MYC expression for samples with HPV integrations on CASC8 and PVT1 and samples without these integration sites; see PVT1 have a significantly higher MYC expression (P = 0.0089). MYC is an oncogene contributing to the development of many human cancersPVT1 near MYC particularly increases its expression in genital cancers and provides one mechanistic aspect of how HPV integrations cause cancer. In addition, the current study shows that HPV16 (8 samples), HPV18 (6 samples) and HPV45 (2 samples) can all integrate into this region. The second hotpot of recurrent virus integration is at the RAD51B locus in 8 CESC samples, which include 3 samples with HPV16, 2 samples with HPV39, one with HPV18 and one with HPV45; interestingly, two HNSC tumors also harbor HPV16 integration at RAD51B. Whole-genome sequencing analysis reveals that HPV integration amplifies the somatic copy number of this regionERBB2 on chromosome 17q12. The integration sites come from HPV16, which is again consistent with previous findingsLINC00393, including HPV16 (two samples), HPV18 (one sample), and HPV45 (two samples). Recurrent HPV integrations at RAD51B, ERBB2 and LINC00393 have also been previously describedERBB2 locus showed significantly increased expression across all exons   . In addihe exons . On the egration . The expPTPN13 in three CESC samples (PTPN13 is a tyrosine phosphatase (PTP) enzyme that is involved in control of cell growth, proliferation, differentiation and transformationPTPN13 lossPTPN13. In order to study the post-transcriptional effect of virus integration, we calculated reads mapped per kilobase per million mapped reads (RPKM) for the exons in PTPN13 and compared the exon-level expression between integration positive cases and the mean of samples without virus integration. As shown in PTPN13 in sample EK-A2PK, while HPV16 integrates at exons 2 and 14 for samples WL-A834 and VS-A8QC, respectively. The virus genes involved in these integration sites are E1, E4, E5, E6 and E7; see PTPN13.We also found new recurrent integration sites at  samples . PTPN13 TERT, and KMT2B (a.k.a. MLL4)) with recurrent HBV integration. TERT and KMT2B are already known in this context from whole-genome sequencingTERT and KMT2B. For example, TERT was recently implicated by somatic events or viral integration in hepatocarcinogensisKMT2B are between exon 3 and exon 8 and integrations often lead to increased expression of exons following these sites; this holds true for TERT, as well . However, abundance in LGG, KIRC, COAD, READ, and LIHC is relatively low (between 102 and 103). We also observed instances having RPHM < 100 , kidney renal clear cell carcinoma (KIRC), COAD, READ, and LIHC. HPV subtypes in BLCA are HPV45, HPV51, HPV56 and HPV6 and virus abundance in four samples is especially high  were observed in tumors, but none in adjacent normal samples. The current study shows that the HBV integration site is a key factor for driving tumorigenesis. The HBV genes may be expressed to sustain infection, but not required for initiation and/or progression of cancer. Lower tumor expression may also be related to evading host immune system response.We compared HBV virus abundance in tumor and paired adjacent normal samples of a large LIHC sample set, failing to find virus enrichment in the tumor, in contrast to the findings for HPV16 in HNSC and CESC, which may represent two different mechanisms of virus-associated tumorigenesis: First, there is a direct carcinogenesis such as what is observed for HPV, in which the virally infected cells directly undergo malignant transformation. The second is the “hit-and-run” scenario where the HBV or HCV-infection triggers inflammation that in turn leads to malignant transformation of neighboring cells without the presence of viral infection. In addition, we found the EBER-1 in HHV4 and RNA2.7 in HHV5 are highly expressed across different tumor samples, supporting likely classifications as virus oncogenes. In addition, the analyses of virus variants in tumor samples reveal the association of virus variants and ethnicity groups for HBV in LIHC and HHV4 in STAD. Finally, we want to note that the current study focuses on known viruses and their current presence on the tumor samples. There are potential novel viruses awaiting discovery, which may also play important roles on the initialization and progression of tumors.HHV4 has been previously shown to be associated with different cancer types, including Hodgkin’s lymphoma, Burkitt’s lymphoma, nasopharyngeal carcinomahttps://cghub.ucsc.edu) across 23 cancer types including esophageal cancer (ESCA), stomach, colon, and rectal adenocarcinomas , liver hepatocellular carcinoma (LIHC), pancreatic adenocarcinoma (PAAD), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), bladder urothelial carcinoma (BLCA), prostate adenocarcinoma (PRAD), uterine carcinosarcoma (UCS), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), head/neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), thyroid carcinoma (THCA), diffuse large B-cell lymphoma (DLBC), acute myeloid leukemia (LAML), sarcoma (SARC), skin cutaneous melanoma (SKCM), glioblastoma multiforme (GBM) and brain lower grade glioma (LGG).We collected 7,372 TCGA RNA-Seq data sets from CGHub  and used CD-HIThttp://www.repeatmasker.org) was used to mask interspersed repeats and low complexity DNA sequences. A sequence failed the quality control criteria if it does not contain a stretch of at least 40 consecutive non-“N” nucleotides  or if greater than 40% of the total length of the sequence is masked . These sequences were removed from further analysis.Further filter human sequences by using MegaBlast against human genome and transcript database.Run MegaBlast against NCBI NT database for the remaining reads after filtering human sequences.Use NCBI taxonomy database to classify viruses and BLAST results to get the specific viral species.The outline of the VirusScan pipeline is illustrated in The VirusScan pipeline is written in Perl and incorporates many standard software packages, such as BWA, RepeatMasker, and the NCBI BLAST suite. The pipeline is fully automated and can run multiple jobs in parallel on a high performance compute cluster, developing from VirusHunter pipeline. In contrast to VirusHunter2963646566672 and the other is RPHM ≥ 103. The observed two clusters may represent two sets of different samples, i.e., one includes samples with low-level HPV contamination or in latent stage of HPV infection and the other consists of samples with actively transcribed HPV. The bimodal distribution suggests that RPHM ≥ 102 is a reasonable cut-off for defining virus-positive samples.With a large collection of cancer samples having high prevalences of HPV, such as HNSC, CESC and BLCA, etc., we have the power to examine the distribution of HPV abundance across all tumor samples. For estimating viral gene expressions and virus integrations, we created a custom reference sequence consisting of the GRCh37 human reference, together with virus types identified during RNA-Seq processing, and realigned all RNA-Seq data to this reference using BWA.Virus gene expression was assessed. Virus gene annotations were downloaded from NCBI. Sequences were aligned against the viral references using BWA and read counts for the targeted viral genes were obtained. Read counts were scaled to report the number of viral reads per 100 million total sequence reads.Extracted pair-end reads from imported bam files, in which reads are aligned to human sequence.Use “BWA sampe” to align the extracted pair-end reads to human plus virus reference. The aligned files were evaluated for presence of human-virus discordant reads, where one read of a read pair maps to human, the other to virus. Such discordant read pair clusters correspond to break points and likely viral integration sites.Run PindelLook *RP file from Pindel output directory for putative integration sites; see Intersect with Ensembl 37.75 gene annotation file and select host genes with ≥10 supporting discordant human-virus pairs for recurrent virus-host integration analysis at gene level see .We use BWA and Pindel to identify the putative virus integration sites. The detailed steps are as following:9 * R)/(N * L) was calculated where R is the number of raw read mapped to the exon and N is the total mapped reads and L is the length of the exon.We generated RPKM value for the exons of different genes based on the TCGA RNA-Seq bams. The detailed steps are as follows. First, bed files were generated for the exon boundary based on Ensembl 37.75 database, followed by the use of the “bedtools -multicov –bam input.bam –bed input.bed” command to count raw reads for all exons. In the final step RPKM based on RPKM = (10http://evolution.genetics.washington.edu/phylip.html) to construct phylogenetic trees based on variant allele fraction for HBV and HHV4. We eliminated duplicate samples, as PHYLIP does not allow such processing. Each analysis used random input of the order of the samples. Output in NEWICK format was used in iTOL (itol.embl.de) to visualize results.We used the contml tool from the PHYLIP toolkit .


L only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIP  •c-FLIP isoforms (L/S) do not directly compete with caspase-8 for binding to FADD•c-FLIP binds to the DISC via a co-operative hierarchical caspase-8-dependent process•LCo-operative and hierarchical binding crucially explains the dual function of c-FLIP•Our unified model defines how c-FLIP isoforms differentially direct cell fate L/S recruitment to FADD is indirect and requires caspase-8. This co-operative hierarchical binding process explains the conundrum of the dual role of c-FLIPL and defines how c-FLIPL/S differentially control cell fate.It is unclear how c-FLIP isoforms can differentially regulate caspase-8 activation to direct cell fate. Hughes et al. show that c-FLIP Apoptotic cell death, which plays a fundamental role during development and homeostasis of multicellular organisms, is orchestrated by the caspase family of cysteine proteases. Deregulated apoptosis is a hallmark of several diseases, including autoimmunity, neurodegeneration, and cancer. The extrinsic apoptotic pathway is initiated by “death ligand”-induced ligation of death receptors (DR), such as CD95 (Fas/Apo1), TRAIL (TNF-related apoptosis-inducing ligand) receptors-1/-2, (TRAIL-R1/R2) and tumor necrosis factor (TNF) receptor-1 (TNF-R1), which form part of the TNFR superfamily . StimulaL) and c-FLIP short (c-FLIPS)  . c-FLIPSthe DISC . While cnecrosis . Likewisrodimers . Thus, iS and overexpressed c-FLIPL can block DISC-dependent procaspase-8 activation  inhibit DISC activation by blocking formation of DED-mediated procaspase-8 oligomers. Significantly, this alternative mechanism for c-FLIPL/S recruitment to the DISC and regulation of DED:DED interactions critically determines procaspase-8 function. We now propose a co-operative and hierarchical binding model that explains the conundrum of the dual functionality of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate. This unified model represents a paradigm shift in terms of our understanding of how c-FLIP regulates the DISC as well as other caspase-8-dependent signaling platforms involved in development, physiology, and disease.We now reveal a hitherto unknown mechanism that explains how c-FLIP35S-labeled procaspase-8  bound to glutathione beads, full-length recombinant FADD (r-FADD), and procaspase-8b (procaspase-8). In this model system, procaspase-8 cleavage to its signature fragments and IETDase activity were only detected in the presence of TRAIL-R1/R2-IcD, r-FADD, and aspase-8 A and S1Aactivity B. These We then investigated the mechanism of procaspase-8 activation in the TRAIL-R1/R2 DISC using a cleavage bioassay for exogenous DISC substrates essential for receptor-mediated apoptosis, namely procaspase-3 and Bid. Both procaspase-3 (C163A) and Bid were efficiently cleaved by a DISC reconstituted with wild-type (WT) procaspase-8 A. MoreovL is also a substrate for active caspase-8, which cleaves c-FLIPL at D376, generating a DED containing p43 fragment and a C-terminal p12 fragment . c-FLIPL was cleaved to its signature p43/p12 fragments by both activated WT and non-cleavable (Quadruple) procaspase-8, but not by an active site (C360A) or dimerization (T467D) mutant  mutant B, demonscleavage . Thus, prization B, schemeL and c-FLIPS function as anti-apoptotic regulators  did not activate procaspase-8    completeaspase-8 C, lane 5the DISC D, schemeL/S competes with procaspase-8 for binding to FADD. To assess binding of procaspase-8 and c-FLIPL/S, we reconstituted the r-DISC using excess levels of catalytically inactive (C360A) procaspase-8. Strikingly, co-incubation with increasing amounts of c-FLIPL/S did not affect procaspase-8 recruitment to FADD. Indeed, despite the presence of procaspase-8, c-FLIPL/S recruitment increased in a concentration-dependent manner , failed to displace procaspase-8 from FADD, and both c-FLIPL and c-FLIPS were recruited to the pre-assembled complex . Remarkably, without procaspase-8, there was negligible recruitment of c-FLIPL/S to FADD. However, increasing amounts of procaspase-8 resulted in concentration-dependent recruitment of both c-FLIPL/S and procaspase-8 , recruited only minimal amounts of c-FLIPS  D. Striki lysates D and S2DTo investigate the co-operative DISC recruitment of c-FLIP and procaspase-8, we first needed to characterize how procaspase-8 is recruited to FADD. A critical phenylalanine/leucine (F122/L123) hydrophobic motif in procaspase-8 DED2 has previously been shown to occupy a hydrophobic pocket in DED1 of an adjacent procaspase-8 molecule  and resuS, namely DED1 pocket residue (H7) and DED2 FL motif (F114/L115)  slightly reduced, but did not abolish, c-FLIPS binding to FADD  was not impaired in the presence of procaspase-8, with binding of c-FLIPS DED1 pocket mutant comparable to that of WT c-FLIPS  A and S4A to FADD B, lane 1FL motif B, lane 2 to FADD , procaspolecules B. Intrigthe DISC B. These S (minus/plus procaspase-8). When compared with WT FADD, mutation of FADD hydrophobic pocket (H9D) had minimal impact on r-DISC recruitment of procaspase-8 . Thus, mutation of the H9 residue in the FADD DED pocket blocks c-FLIPL/S recruitment, but addition of procaspase-8 restores c-FLIPL/S recruitment via procaspase-8:c-FLIP DED-mediated interactions .Our reconstituted DISC experiments fully recapitulated the suggested biphasic effects of c-FLIPfunction . Thus, caspase-8 . c-FLIPL c-FLIPL  and 2. Tocessing . Thus, lthe DISC . As leveive DISC . Thus, pS is a potent inhibitor of caspase-8 as it results in concentration-dependent inhibition of DISC-mediated procaspase-8 activation  and led us to hypothesize that procaspase-8:c-FLIPS heterodimers are catalytically inactive and moreover inhibit DISC signaling by modulating DED-mediated caspase-8 oligomerization. Our observation that only a finite amount of c-FLIPS is recruited to a reconstituted DISC   . This alL and c-FLIPS to inhibit procaspase-8 activation. Thus, c-FLIPS potently blocks procaspase-8 processing, but very high levels of c-FLIPL are required to inhibit caspase-8  plus/minus c-FLIPL/S, for 16 hr at 20°C (unless indicated otherwise). Control reconstitutions contained beads coated with GST alone. Bead-associated complexes were analyzed by SDS-PAGE/western blotting. r-DISC-associated caspase-8 activity was measured by cleavage of the fluorogenic substrate Ac-IETD.AFC or by bioassay using recombinant procaspase-3 active-site mutant (C163A) or Bid  or Bid .S-expressing BJAB cells as described (S-expressing keratinocytes (HaCaT cells) as described  for 60 min at 4°C. DISC complexes were analyzed by western blotting or mass spectrometry.Biotin-labeled TRAIL (bTRAIL) was generated and TRAIL DISC affinity purified from control or c-FLIPescribed . CD95 DIescribed , with thLC-MS/MS was used to identify and quantify DISC proteins see . BrieflyHeLa, MCF-7, or HEK293 cells were seeded onto coverslips (coated with gelatin or poly-D-Lysine) 24 hr prior to transfection. Cells were transfected in the presence of the pan-caspase inhibitor, zVAD.FMK (50 μM), and fixed as described ; EGFP alQ14790) was generated as described (O15519) were submitted to the Phyre2 server, and the structure resulting from threading of this sequence through the published structure of MC159 (PDB: 2BBR) was selected as our modeled structure of c-FLIP DED1/2. The DED domains of caspase-8, c-FLIP, and FADD (PDB: 2GF5) were aligned using LSQman. Pymol (www.pymol.org) was used to generate structural models.The modeled structure of the DEDs of caspase-8 (amino acids 1–183; UniProtKB: escribed . Amino aM.M., M.A.H., and K.C. conceived the study, designed experiments, and analyzed results. M.A.H. carried out the majority of experiments; I.R.P., confocal microscopy; R.J.-J., LC-MS/MS; S.H. and M.F., c-FLIP overexpression; L.F. and J.W.R.S., structural modeling. M.M., M.A.H., M.L., and K.C. wrote the manuscript with input from all co-authors.