UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription.

More than half the world's population is infected with human cytomegalovirus (HCMV), causing congenital birth defects and impacting the immuno-compromised. Many of the >170 HCMV genes remain uncharacterized, and this gap in knowledge limits the development of novel antivirals. In this study, we investigated the essential viral protein UL49 and found it displayed leaky late expression kinetics, and localized to nuclear replication compartments. Cells infected with mutant UL49 virus were unable to produce infectious virions and phenocopied other beta-gamma viral pre-initiation complex (vPIC) subunit (UL79, UL87, UL91, UL92, and UL95) mutant infections. RNA-seq analysis of vPIC mutant infections revealed a consistent diminution of genes encoding capsid subunits, including TRX2/UL85 and MCP/UL86, envelope glycoproteins gM, gL and gO, and egress-associated tegument proteins UL99 and UL103. Therefore, as a member of the vPIC, UL49 serves as a fundamental HCMV effector that governs viral gene transcription required to complete the replication cycle.

Induced endothelial cells from peripheral arterial disease patients and neonatal fibroblasts have comparable angiogenic properties.

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.

Herpesviruses hijack host exosomes for viral pathogenesis.

Herpesviruses are remarkable pathogens possessing elaborate mechanisms to seize various host cellular components for immune evasion, replication, and virion egress. As viruses are dependent upon their hosts, investigating this intricate interplay has revealed that the exosome pathway is utilised by alpha (Herpes Simplex Virus 1), beta (Human Cytomegalovirus, and Human Herpesvirus 6) and gamma (Epstein-Barr Virus, and Kaposi Sarcoma-associated Herpesvirus) herpesviruses. Virions and exosomes share similar properties and functions. For example, exosomes are small membranous nanovesicles (30-150nm) released from cells that contain proteins, DNA, and various coding and non-coding RNA species. Given exosomes can shuttle various molecular cargo from a donor to recipient cell, they serve as important vehicles facilitating cell-cell communication. Therefore, exploitation by herpesviruses impacts several aspects of infection including: i) acquisition of molecular machinery for secondary envelopment and viral assembly, ii) export of immune-related host proteins from infected cells, iii) enhancing infection in surrounding cells via transfer of viral proteins, mRNA and miRNA, and iv) regulation of viral protein expression to promote persistence. Studying the dichotomy that exists between host exosomes and herpesviruses has two benefits. Firstly, it will reveal the precise pathogenic mechanisms viruses have evolved, generating knowledge for antiviral development. Secondly, it will shed light upon fundamental exosome characteristics that remain unknown, including cargo selection, protein trafficking, and non-canonical biogenesis.

A comparison of the pro-angiogenic potential of human induced pluripotent stem cell derived endothelial cells and induced endothelial cells in a murine model of peripheral arterial disease.

BACKGROUND: Endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) promote angiogenesis, and more recently induced endothelial cells (iECs) have been generated via fibroblast trans-differentiation. These cell types have potential as treatments for peripheral arterial disease (PAD). However, it is unknown whether different reprogramming methods produce cells that are equivalent in terms of their pro-angiogenic capabilities. OBJECTIVES: We aimed to directly compare iPSC-ECs and iECs in an animal model of PAD, in order to identify which cell type, if any, displays superior therapeutic potential. METHODS: IPSC-ECs and iECs were generated from human fibroblasts, and transduced with a reporter construct encoding GFP and firefly luciferase for bioluminescence imaging (BLI). Endothelial phenotype was confirmed using in vitro assays. NOD-SCID mice underwent hindlimb ischaemia surgery and received an intramuscular injection of either 1×106 iPSC-ECs, 1×106 iECs or control vehicle only. Perfusion recovery was measured by laser Doppler. Hindlimb muscle samples were taken for histological analyses. RESULTS: Perfusion recovery was enhanced in iPSC-EC treated mice on day 14 (Control vs. iPSC-EC; 0.35±0.04 vs. 0.54±0.08, p<0.05) and in iEC treated mice on days 7 (Control vs. iEC; 0.23±0.02 vs. 0.44±0.06, p<0.05), 10 (0.31±0.04 vs. 0.64±0.07, p<0.001) and 14 (0.35±0.04 vs. 0.68±0.07, p<0.001) post-treatment. IEC-treated mice also had greater capillary density in the ischaemic gastrocnemius muscle (Control vs. iEC; 125±10 vs. 179±11 capillaries/image; p<0.05). BLI detected iPSC-EC and iEC presence in vivo for two weeks post-treatment. CONCLUSIONS: IPSC-ECs and iECs exhibit similar, but not identical, endothelial functionality and both cell types enhance perfusion recovery after hindlimb ischaemia.

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells.

INTRODUCTION: Recent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells. METHODS: Human coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug's peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined. RESULTS: Both drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs. CONCLUSION: We demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.

A study of the virulence in mice of high copying fidelity variants of human enterovirus 71.

Polioviruses with a G64S mutation in the 3D polymerase have enhanced replication fidelity and are attenuated in animal models. Here we describe the mouse virulence properties of high replication fidelity 3D polymerase variants of human enterovirus 71 (HEV71), with mutations at positions 3D-S264L, 3D-G64R or at 3D-S264L plus 3D-G64R. Mouse-adapted strains (MP-G64R, MP-S264L and MP-S264L-G64R) were constructed in order to compare the virulence of the 3D polymerase variants with that of mouse-adapted parental virus (MP-26M). MP-S264L and MP-S264L-G64R were attenuated in mice (mean survival time 7.0 and 7.5 days p.i., respectively) compared to MP-G64R and MP-26M (mean survival time 6.5 and 6.0 days p.i., respectively). MP-26M and MP-G64R infection induced early onset, severe generalised necrotising myositis, whereas MP-S264L and MP-S264L-G64R infection induced a later onset, mild and focal skeletal muscle myositis. Our findings demonstrate that only the 3D-S264L mutation attenuates HEV71 in mice, suggesting that the high replication fidelity phenotype is not essential for virulence attenuation in this model.

Ribavirin-resistant mutants of human enterovirus 71 express a high replication fidelity phenotype during growth in cell culture.

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 µM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 µM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 µM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.

Selection and characterisation of guanidine-resistant mutants of human enterovirus 71.

The replication of human enterovirus 71 (HEV71) in cell culture is inhibited by concentrations of guanidine that do not have an observable adverse effect on host cell metabolism. Although the HEV71 non-structural protein 2C is known to play an important role in viral RNA replication, its precise biochemical activities and structure have not been fully determined. Here we describe amino acid substitutions in HEV71 protein 2C that confer resistance to guanidine. Three guanidine-resistant virus populations were independently isolated and found to contain five mutations in protein 2C, one of which, A4657T (2C-M193L), was present in two of the independently selected populations. This mutation was introduced into a HEV71 infectious cDNA clone and was sufficient to confer complete resistance to growth inhibition in the presence of 4mM guanidine. In the first guanidine-resistant population selected, the 2C-M193L mutation occurred in association with an additional mutation, A4459G (2C-I127V), located in the putative cis-acting replication element (cre) of coding region 2C. This mutation conferred only partial guanidine resistance when introduced into the HEV71-26M infectious clone. When the 2C-I127V and 2C-M193L mutations were introduced into HEV71-26M together, the 2C-I127V mutation did not increase the level of guanidine resistance due to the 2C-M193L mutation alone. This study confirms that guanidine resistance can be readily selected in HEV71 and is attributable to mutations within protein 2C.

Environmental surveillance of Non-Polio Enteroviruses in Iran.

BACKGROUND: Enteroviruses can shed in feces for several weeks, so many excrete viruses can remain infectious for a long time in environment. Therefore, by detecting enteroviruses in environmental specimens and sewage, we can understand this virus circulation, the approximate ratio of contaminated persons in society and they are suitable indicators for environmental surveillance. METHODS: Since March 2006 to February 2007, 86 specimens from Sistan & Balouchestan, 63 specimens from Tehran and 48 samples from Fars sewage disposal systems and surface water were collected by Grab Sample method and tested for enteroviruses directly by using two concentration methods: Pellet and Two-phase. Then Non-Polio Enteroviruses (NPEV) were serotyped by microneutralization method. RESULTS: Enteroviruses were isolated from 49(56.98%) of specimens in Sistan & Baluchestan,38(60.32%) in Tehran and 11(22.92%) in Fars. Besides, the majority of Non-Polio Enteroviruses related to Non-typable Enteroviruses (N.T.E.V), E11 (31.52%), COX-B (27.58%), E7 (17.73%) and E4 (21.67%). CONCLUSION: Environmental surveillance has been used successfully in monitoring enteric virus circulation and assessing the extent or duration of epidemic non polioviruses in specific populations. The results of this research show the seasonal circulation of enteroviruses in different parts of Iran.