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Alterations in cell cycle regulators are common in hepatocellular carcinoma (HCC). We tested the efficacy of composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib on HCC. In vitro, dinaciclib exhibited potent antiproliferative activities in HCC cell lines regardless of Rb or c-myc expression levels. Dinaciclib significantly downregulated the phosphorylation of Rb (target of CDKs 1 and 2), ataxia telangiectasia mutated kinase (target of CDK5), and RNA polymerase II (target of CDK9) in the HCC cells. In xenograft studies, mice receiving dinaciclib tolerated the treatment well without significant body weight changes and exhibited a significantly slower tumor growth rate than the mice receiving vehicles. RNA interference (RNAi) of CDKs 1 and 9 was more effective in inhibiting the cell proliferation of HCC cells than RNAi of CDKs 2 and 5. Overexpression of CDK9 significantly reduced the efficacy of dinaciclib in HCC cells, but overexpression of CDK1 did not. In conclusion, composite inhibition of CDKs 1, 2, 5, and 9 through dinaciclib exhibited potent in vitro and in vivo activity against HCC. CDK9 inhibition might be the crucial mechanism. [ABSTRACT FROM AUTHOR]

Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses has been reported. We evaluated cell lines commonly used to isolate viruses and diagnose related diseases for their susceptibility to SARS-CoV-2. Although multiple kidney cell lines from monkeys were susceptible to SARS-CoV-2, we found many cell types derived from humans, dogs, minks, cats, mice, and chicken were not. We analyzed MDCK cells, which are most commonly used for surveillance and study of influenza viruses, and found that they were not susceptible to SARS-CoV-2. The low expression level of the angiotensin converting enzyme 2 receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by overexpression of canine angiotensin converting enzyme 2 in trans, strengthened the cellular barrier to productive infection. Moreover, a D614G mutation in the spike protein did not appear to affect SARS-CoV-2 cell tropism. Our findings should help avert inadvertent propagation of SARS-CoV-2 from diagnostic cell lines. [ABSTRACT FROM AUTHOR]

We previously produced a replication-defective West Nile virus lacking NS1 WNV-ΔNS1) that could propagate at low levels (105 IU/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-ΔNS1 as a non-infectious vaccine. To explore this idea, we developed an NS1-expressing Vero cell line (VeroNS1) that significantly improved the yield of WNV-ΔNS1 (108 IU/ml). We evaluated the safety and efficacy of WNV-ΔNS1 in mice. WNV-ΔNS1 appeared to be safe as no replicative virus on naïve Vero cells after continuously culturing of WNV-ΔNS1 on VeroNS1 cells for fifteen rounds. WNV-ΔNS1 was non-infectious in mice, even when IFNAR-/- mice were administrated with a high dose of WNV-ΔNS1. A single dose of WNV-ΔNS1 vaccination protected mice from a highly lethal challenge of WT WNV. The antibody response against WNV correlated well with the protection of vaccinated mice. Our study demonstrates the potential of the NS1 trans complementation system as a new platform for flavivirus vaccine development. [ABSTRACT FROM AUTHOR]

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10[sup 9] VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10[sup 10] VLPs per 10[sup 6] transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus, tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8[sup +]-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 10[sup 7] VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10[sup 6] splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses. [ABSTRACT FROM AUTHOR]