The Bovine Lactophorin C-Terminal Fragment and PAS6/7 Were Both Potent in the Inhibition of Human Rotavirus Replication in Cultured Epithelial Cells and the Prevention of Experimental Gastroenteritis

An Assay for Detecting Neutralization of Rotavirus Infection by Quantitative Determination of VP6 Protein Fluorescence Intensity

“…3A and B). This is consistent with a previous report by Inagaki et al 8) In comparing the focus reduction assay and the new method, the results indicated that the MICs of the rota whey were 0.7 mg/mL (Fig. 3A) and 1.3 mg/mL (Fig.…”

“…1) Consequently, researchers have continued to investigate safe and effective inhibitors of infection, and many previous studies have suggested compounds to prevent infection. [6][7][8][9] Previously published rotavirus infection studies used the focus reduction assay extensively, is a method that involves staining infected cells with a fluorescenceconjugated antibody and counting the stained cells by means of fluorescence microscopy. For example, the embryonic rhesus monkey kidney epithelial MA104 cell line was inoculated with a mixture of sample protein and virus, the infected cells were stained by indirect immunoantibody assay, and they were finally examined microscopically to identify immunoperoxidase-stained foci, which indicate infection.…”

“…For example, the embryonic rhesus monkey kidney epithelial MA104 cell line was inoculated with a mixture of sample protein and virus, the infected cells were stained by indirect immunoantibody assay, and they were finally examined microscopically to identify immunoperoxidase-stained foci, which indicate infection. [7][8][9] A major limitation of this method is that it fails to provide information about the underlying cells. Furthermore, to do a focus reduction assay, considerable skill is required for accurate discrimination of infected cells from noninfected cells, which typically takes a long time to perform since it relies on manual counting.…”

“…The rotavirus MO strain, serotype G3P [8], was activated with trypsin (SigmaAldrich St. Louis, MO) and added into MA104 cells, as described previously. 7) Rotavirus VP6 of the infected cell was stained with a combination of the PO-13 monoclonal anti-pigeon rotavirus antibody and FITCconjugated goat anti-mouse IgG serum (American Qualex, San Clemente, CA).…”

“…8) For the new method, after treatment with trypsin, making a virus dilution of 1:8 with EMEM containing 2% FCS, we mixed it with an equal volume (200 mL) of two serially diluted samples in microtubes, and incubated them for 1 h at 37 C. The various mixtures of samples with virus were administered (100 mL) to each well containing a confluent monolayer of MA104 cells. As a control, we also included wells with only the virus for infection.…”