Genome sequences of the 15 bluetongue virus vaccine strains incorporated in the South African live-attenuated vaccine.

Bluetongue disease in endemic areas is predominantly controlled through vaccination with live-attenuated vaccines. Sequencing of the original master seed viruses used in the production of Onderstepoort Biological Products vaccine was conducted. Nucleotide identities of 82.97%-100% were obtained for all sequences when compared to South African reference strains.

Investigating the Role of African Horse Sickness Virus VP7 Protein Crystalline Particles on Virus Replication and Release.

African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.

Transcriptomics Advancement in the Complex Response of Plants to Viroid Infection.

Viroids are the smallest plant pathogens, consisting of a single-stranded circular RNA of less than 500 ribonucleotides in length. Despite their noncoding nature, viroids elicit disease symptoms in many economically important plant hosts, and are, thus, a class of pathogens of great interest. How these viroids establish disease within host plants, however, is not yet fully understood. Recent transcriptomic studies have revealed that viroid infection influences the expression of genes in several pathways and processes in plants, including defence responses, phytohormone signalling, cell wall modification, photosynthesis, secondary metabolism, transport, gene expression and protein modification. There is much debate about whether affected pathways signify a plant response to viroid infection, or are associated with the appearance of disease symptoms in these interactions. In this review, we consolidate the findings of viroid-host transcriptome studies to provide an overview of trends observed in the data. When considered together, changes in the gene expression of different hosts upon viroid infection reveal commonalities and differences in diverse interactions. Here, we discuss whether trends in host gene expression can be correlated to plant defence or disease development during viroid infection, and highlight avenues for future research in this field.

Efficacy of SAT2 Foot-and-Mouth Disease Vaccines Formulated with Montanide ISA 206B and Quil-A Saponin Adjuvants.

The effective control of foot-and-mouth disease (FMD) relies strongly on the separation of susceptible and infected livestock or susceptible livestock and persistently infected wildlife, vaccination, and veterinary sanitary measures. Vaccines affording protection against multiple serotypes for longer than six months and that are less reliant on the cold chain during handling are urgently needed for the effective control of FMD in endemic regions. Although much effort has been devoted to improving the immune responses elicited through the use of modern adjuvants, their efficacy is dependent on the formulation recipe, target species and administration route. Here we compared and evaluated the efficacy of two adjuvant formulations in combination with a structurally stabilized SAT2 vaccine antigen, designed to have improved thermostability, antigen shelf-life and longevity of antibody response. Protection mediated by the Montanide ISA 206B-adjuvanted or Quil-A Saponin-adjuvanted SAT2 vaccines were comparable. The Montanide ISA 206B-adjuvanted vaccine elicited a higher SAT2 neutralizing antibody response and three times higher levels of systemic IFN-γ responses at 14- and 28-days post-vaccination (dpv) were observed compared to the Quil-A Saponin-adjuvanted vaccine group. Interestingly, serum antibodies from the immunized animals reacted similarly to the parental vaccine virus and viruses containing mutations in the VP2 protein that simulate antigenic drift in nature.

Genetic Basis of Antigenic Variation of SAT3 Foot-And-Mouth Disease Viruses in Southern Africa.

Foot-and-mouth disease (FMD) continues to be a major burden for livestock owners in endemic countries and a continuous threat to FMD-free countries. The epidemiology and control of FMD in Africa is complicated by the presence of five clinically indistinguishable serotypes. Of these the Southern African Territories (SAT) type 3 has received limited attention, likely due to its restricted distribution and it being less frequently detected. We investigated the intratypic genetic variation of the complete P1 capsid-coding region of 22 SAT3 viruses and confirmed the geographical distribution of five of the six SAT3 topotypes. The antigenic cross-reactivity of 12 SAT3 viruses against reference antisera was assessed by performing virus neutralization assays and calculating the r1-values, which is a ratio of the heterologous neutralizing titer to the homologous neutralizing titer. Interestingly, cross-reactivity between the SAT3 reference antisera and many SAT3 viruses was notably high (r1-values >0.3). Moreover, some of the SAT3 viruses reacted more strongly to the reference sera compared to the homologous virus (r1-values >1). An increase in the avidity of the reference antisera to the heterologous viruses could explain some of the higher neutralization titers observed. Subsequently, we used the antigenic variability data and corresponding genetic and structural data to predict naturally occurring amino acid positions that correlate with antigenic changes. We identified four unique residues within the VP1, VP2, and VP3 proteins, associated with a change in cross-reactivity, with two sites that change simultaneously. The analysis of antigenic variation in the context of sequence differences is critical for both surveillance-informed selection of effective vaccines and the rational design of vaccine antigens tailored for specific geographic localities, using reverse genetics.

Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the ßD-ßE loop and 110-112 in the ßF-ßG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

Pathogenesis, biophysical stability and phenotypic variance of SAT2 foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals, which severely decreases livestock productivity. FMD virus (FMDV), the causative agent, initiates infection by interaction with integrin cellular receptors on pharyngeal epithelium cells, causing clinical signs one to four days after transmission to a susceptible host. However, some Southern African Territories (SAT) viruses have been reported to cause mild or subclinical infections that may go undiagnosed in field conditions and are likely to be more common than previously expected. The studies presented here demonstrate that not all SAT2 viruses are equally virulent in cattle. The two SAT2 viruses, ZIM/5/83 and ZIM/7/83, were both highly attenuated in cattle, as evidenced by the mild clinical signs observed after needle challenge, while two incongruent SAT2 viruses showed significantly different clinical signs in challenged cattle. We then explored the ability of the SAT2 viruses to infect different cell types with defined receptors that are utilised by FMDV and found differences in their ability to lyse cells in culture and to compete in a controlled cell culture environment. The population sequence variation between ZIM/5/83 and ZIM/7/83 revealed multiple sites of single nucleotide variants of low frequency between the predominant virus populations, as could be expected from the genome of an RNA virus. An assessment of the biophysical stability of SAT2 virions during acidification indicated that the SAT2 virus EGY/09/12 was more resilient to acidification than the ZIM/5/83 and ZIM/7/83 viruses; however, whether this difference relates to differences in virulence in vivo is unclear. This study is a consolidated view of the key findings of SAT2 viruses studied over a 14-year period involving many different experiments.

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Production of foot-and-mouth disease virus SAT2 VP1 protein.

The seven serotypes of foot-and-mouth disease virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L-1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing virus as reagent, or having to raise reagent antibodies.

Targeted mutational analysis to unravel the complexity of African horse sickness virus NS3 function in mammalian cells.

The African horse sickness virus non-structural protein 3 (NS3) is involved in the final stages of infection. To gain insight into the function of different NS3 domains, we generated reverse genetics-derived mutants, each expressing a modified version of the protein. A functional comparison of these mutants to the wild-type virus in mammalian cells indicated the variable contribution of the different domains to the cytopathic effect and in ensuring effective virus trafficking and release. The transmembrane domains were determined as essential mediators of NS3 localisation, as the abnormal processing of these mutant proteins resulted in their nuclear localisation and interaction with NS1. NS3 cytoplasmic domain disruptions resulted in increased cytosolic virus particle accumulation and abnormal virion tethering to plasma membranes. Other aspects of infection were also affected, such as VIB formation and distribution of the outer capsid proteins. Overall, these results illustrate the intricate role of NS3 in the infection cycle.

Inherent biophysical stability of foot-and-mouth disease SAT1, SAT2 and SAT3 viruses.

Foot-and-mouth disease (FMD) virus (FMDV) isolates show variation in their ability to withstand an increase in temperature. The FMDV is surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot climate regions where FMD is prevalent. The three SAT serotypes, with their particularly low biophysical stability also only yield vaccines of low protective capacity, even with multiple booster vaccinations. The aim of the study was to determine the inherent biophysical stability of field SAT isolates. To characterise the biophysical stability of 20 SAT viruses from Southern Africa, the thermofluor assay was used to monitor capsid dissociation by the release of the RNA genome under a range of temperature, pH and ionic conditions. The SAT2 and SAT3 viruses had a similar range of thermostability of 48-54 °C. However, the SAT1 viruses had a wider range of thermostability with an 8 °C difference but with many viruses being unstable at 43-46 °C. The thermostable A-serotype A24 control virus had the highest thermostability of 55 °C with some SAT2 and SAT3 viruses of similar thermostability. There was a 10 °C difference between the most unstable SAT virus (SAT1/TAN/2/99) and the highly stable A24 control virus. SAT1 viruses were generally more stable compared to SAT2 and SAT3 viruses at the pH range of 6.7-9.1. The effect of ionic buffers on capsid stability showed that SAT1 and SAT2 viruses had an increased stability of 2-9 °C and 2-6 °C, respectively, with the addition of 1 M NaCl. This is in contrast to the SAT3 viruses, which did not show improved stabilisation after addition of 1 M or 0.5 M NaCl buffers. Some buffers showed differing results dependent on the virus tested, highlighting the need to test SAT viruses with different solutions to establish the most stabilising option for storage of each virus. This study confirms for the first time that more stable SAT field viruses are present in the southern Africa region. This could facilitate the selection of the most stable circulating field strains, for adaptation to cultured BHK-21 cells or manipulation by reverse genetics and targeted mutation to produce improved vaccine master seed viruses.

Influence of the ferric uptake regulator (Fur) protein on pathogenicity in Pectobacterium carotovorum subsp. brasiliense.

Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.

The Culicoides sonorensis inhibitor of apoptosis 1 protein protects mammalian cells from apoptosis induced by infection with African horse sickness virus and bluetongue virus.

African horse sickness virus (AHSV) and bluetongue virus (BTV) are arboviruses of the genus Orbivirus that are transmitted to their vertebrate hosts by Culicoides biting midges. These orbiviruses exhibit lytic infection (apoptosis) in mammalian cells, but cause persistent infection with no cytopathic effects in Culicoides sonorensis cells. Although regulation of apoptosis could thus be integral for establishing persistent virus infection in midge cells, nothing is known about the presence and function of apoptosis pathways in Culicoides midges and their derived cell lines. Here, we report the cloning and functional characterization of an inhibitor of apoptosis protein (IAP), designated CsIAP1, from C. sonorensis cells. The CsIAP1 protein contains two baculoviral IAP repeat (BIR) domains and a RING domain. Silencing of the Cs iap1 gene in C. sonorensis cells caused apoptosis, indicating that CsIAP1 plays a role in cell survival. Stable expression of the CsIAP1 protein in BSR mammalian cells suppressed apoptosis induced by AHSV-4 and BTV-10 infection, and biochemical data indicated that CsIAP1 is an inhibitor of mammalian caspase-9, an initiator caspase in the intrinsic apoptotic pathway. Mutagenesis studies indicated that the BIR2 and RING domains are required for the anti-apoptotic activity of CsIAP1. The results suggest that the mechanism by which CsIAP1 suppresses apoptosis in insect cells may involve inhibition of a Culicoides caspase-9 homologue through a mechanism that requires both the BIR2 and RING domains. This study provides the first evidence that the CsIAP1 protein is a key negative regulator of apoptosis in C. sonorensis cells.

A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain.

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus.

In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.

Characterization of two LuxI/R homologs in Pantoea ananatis LMG 2665T.

Quorum sensing (QS) plays an important role in the regulation of bacteria-host interactions and ecological fitness in many bacteria. In this study, 2 luxI/R homologs, namely eanI/eanR and rhlI/rhlR, were identified in the genome sequence of Pantoea ananatis LMG 2665T. To determine a role for these luxI/R homologs in pathogenicity and biofilm formation, mutant bacterial strains lacking either eanI/R or rhlI/R and both of these homologs were generated. The results indicated that both the RhlI/R and EanI/R systems are required for pathogenicity and biofilm formation in strain LMG 2665T. This is the first study to characterize the biological significance of the RhlI/R QS system in P. ananatis.

African horse sickness virus infects BSR cells through macropinocytosis.

Cellular pathways involved in cell entry by African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, have not yet been determined. Here, we show that acidic pH is required for productive infection of BSR cells by AHSV-4, suggesting that the virus is likely internalized by an endocytic pathway. We subsequently analyzed the major endocytic routes using specific inhibitors and determined the consequences for AHSV-4 entry into BSR cells. The results indicated that virus entry is dynamin dependent, but clathrin- and lipid raft/caveolae-mediated endocytic pathways were not used by AHSV-4 to enter and infect BSR cells. Instead, binding of AHSV-4 to BSR cells stimulated uptake of a macropinocytosis-specific cargo and inhibition of Na(+)/H(+) exchangers, actin polymerization and cellular GTPases and kinases involved in macropinocytosis significantly inhibited AHSV-4 infection. Altogether, the data suggest that AHSV-4 infects BSR cells by utilizing macropinocytosis as the primary entry pathway.

Synthesis of empty african horse sickness virus particles.

As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.

Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus.

Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics approaches for engineering the BTV genome rely upon in vitro synthesis of capped RNA transcripts from cloned cDNA corresponding to viral genome segments. In an effort to expand the utility of BTV reverse genetics, we constructed a reverse genetics vector containing a T7 RNA polymerase promoter, hepatitis delta ribozyme sequence and T7 RNA polymerase terminator sequence. Viable virus was recovered following transfection of mammalian cells, expressing T7 RNA polymerase, with 10 plasmid constructs representing the cloned BTV-1 genome. Furthermore, the plasmid-based reverse genetics system was used successfully to isolate viable cross-serotype reassortant viruses and a mutant virus containing a defined mutation in the replicating viral genome. The new reverse genetics platform established here for BTV is likely applicable to other orbiviruses.

Angioseal™ as a hemostatic device for direct carotid puncture during endovascular procedures.

INTRODUCTION: Direct carotid artery puncture (DCP) is employed in patients with tortuous anatomy and peripheral vascular disease when the peripheral arteries are not available for vascular access. Manual compression is the only method of achieving hemostasis following DCP and, till date, the use of a closure device for DCP has been reported in only one patient. In this study we sought to analyze our experience with the use of closure device for DCP. METHODS: This is a retrospective study of patients in whom Angioseal™ was used following DCP for neuroendovascular procedures. Medical charts and imaging of these patients was reviewed for any abnormalities pertaining to the use of the closure device. RESULTS: A total of eight patients were included in the study. Angioseal™ was used in all the patients. There were no complications related to the use of the closure device in any of the eight patients. Immediate post-procedure angiography done in one patient did not show any structural or hemodynamic abnormalities within the carotid artery lumen. At 6 months follow-up imaging, there was no evidence stenosis or vascular wall abnormality in any of the patients. There were no adverse clinical reactions related to the use of closure device. CONCLUSION: In our experience, Angioseal™ may be a safe and off-label effective closure device for patients undergoing DCP for neuroendovascular procedures. It obviates the need for manual compression without causing any structural or hemodynamic changes within the carotid artery. Larger studies with longer follow-up are required to establish its safety in patients undergoing DCP.