A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Apoptotic properties of Citrus sudachi Hort, ex Shirai (Rutaceae) extract on humanA549 and HepG2 cancer cells

Purpose: To investigate whether Citrus sudachi harvested at two stages of maturity can induce toxicity in a cell-specific manner and to determine the possible mechanisms of Citrus sudachi-induced cytotoxic responses in two types of cancer cells (human lung adenocarcinoma A549 and hepatocellular carcinoma HepG2 cells) and two normal cell lines (lung 16HBE140- and liver CHANG cells). Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and annexin V/propidium iodidle assay were used to test the antiproliferative activity and apoptosis of methanol extract of Citrus sudachi, respectively. Griess reaction and reverse transcriptase-polymerase chain reaction (RT-PCR) were carried out to evaluate nitric oxide (NO•) production and the mRNA levels of inhibitors of apoptosis (IAP). Results: Citrus sudachi exerted cytotoxicity in a time-dependent manner in cancer cells which increased with increase in maturity but did not affect normal cells. Citrus sudachi was found to induce accumulation of cells in the sub-G1 cell cycle phase, fragmentation of DNA and cell death with characteristics of apoptosis, in both types of cancer cells. Moreover, Citrus sudachi upregulated cellular NO• produced by activation of nitric oxide synthase (NOS), while it suppressed the levels of IAP mRNA in both types of cancer cells. Conclusion: The results obtained suggest that Citrus sudachi induces apoptosis in A549 and HepG2 cells, which may be mediated by NO•. There is need for further studies on the role of Citrus sudachi in cancer treatment.

Antiviral activity and mechanism of action of arbidol against Hantaan virus infection

Purpose: To investigate the activity and mechanism of action of arbidol against Hantaan virus (HTNV) activity by modulating inflammation via TLR-4 pathway. Methods: HUVEC cells infected with HTNV 76-118 were treated with serially diluted arbidol solutions at -2h (2 h before viral infection, pre-treatment mode), 0 h (at the same time as viral infection, simultaneous treatment mode) or 2 h (2 h after viral infection, post-treatment mode). The transcript levels of TLR4 were detected by semi-quantitative reverse transcription-PCR (RT-PCR) at 6, 12, 18, and 24 h later. The levels of iNOS and TNF-α were examined using enzyme-linked immunosorbent assay (ELISA). Results: Pre-treatment with arbidol, rather than simultaneous treatment or post-treatment, effectively inhibited up-regulation of cellular TLR4 expression (up to 40 ± 6.1 % inhibition) and activity of supernatant iNOS induced by HTNV infection(up to 44.1 ± 9.4 % inhibition), as well as in a LPSstimulated inflammatory endothelial cell. Arbidol decreased the elevated TNF-α levels induced by LPS stimulation. Conclusion: These results are the first evidence that arbidol modulates viral PRRs signaling and its consequential inflammatory cytokine/chemokine response during hantavirus infection.