Inhibitory effects of kaempferol, quercetin and luteolin on the replication of human parainfluenza virus type 2 in vitro.

The eight flavonoids, apigenin, chrysin, hesperidin, kaempferol, myricetin, quercetin, rutin and luteolin were tested for the inhibition of human parainfluenza virus type 2 (hPIV-2) replication. Three flavonoids out of the eight, kaempferol, quercetin and luteolin inhibited hPIV-2 replication. Kaempferol reduced the virus release (below 1/10,000), partly inhibited genome and mRNA syntheses, but protein synthesis was observed. It partly inhibited virus entry into the cells and virus spreading, and also partly disrupted microtubules and actin microfilaments, indicating that the virus release inhibition was partly caused by the disruption of cytoskeleton. Quercetine reduced the virus release (below 1/10,000), partly inhibited genome, mRNA and protein syntheses. It partly inhibited virus entry and spreading, and also partly destroyed microtubules and microfilaments. Luteolin reduced the virus release (below 1/100,000), largely inhibited genome, mRNA and protein syntheses. It inhibited virus entry and spreading. It disrupted microtubules and microfilaments. These results indicated that luteolin has the most inhibitory effect on hPIV-2 relication. In conclusion, the three flavonoids inhibited virus replication by the inhibition of genome, mRNA and protein syntheses, and in addition to those, by the disruption of cytoskeleton in vitro.

Inhibitory effect of traditional herbal (kampo) medicines on the replication of human parainfluenza virus type 2 in vitro.

Thirteen herbal medicines, Kakkonto (TJ-001), Kakkontokasenkyushin'i (TJ-002), Hangekobokuto (TJ-016), Shoseiryuto (TJ-019), Maoto (TJ-027), Bakumondoto (TJ-029), Hochuekkito (TJ-041), Goshakusan (TJ-063), Kososan (TJ-070), Chikujountanto (TJ-091), Gokoto (TJ-095), Saibokuto (TJ-096), and Ryokankyomishingeninto (TJ-119) were tested for human parainfluenza virus type 2 (hPIV-2) replication. Eight (TJ-001, TJ-002, TJ-019, TJ-029, TJ-041, TJ-063, TJ-095 and TJ-119) out of the thirteen medicines had virus growth inhibitory activity. TJ-001 and TJ-002 inhibited virus release, and largely inhibited genome, mRNA and protein syntheses. TJ-019 slightly inhibited virus release, inhibited gene and mRNA syntheses, and largely inhibited protein synthesis. TJ-029 slightly inhibited virus release, largely inhibited protein synthesis, but gene and mRNA syntheses were unaffected. TJ-041 only slightly inhibited virus release, the gene and mRNA syntheses, but largely inhibited protein synthesis. TJ-091 largely inhibited gene, mRNA and protein syntheses. TJ-095 largely inhibited gene synthesis, but NP and HN mRNAs were slightly detected, and protein syntheses were observed. TJ-119 inhibited gene, mRNA and protein syntheses. TJ-001, TJ-002, TJ-091, TJ-095 and TJ-119 inhibited multinucleated giant cell formation derived from cell-to-cell spreading of virus. However, in TJ-019, TJ-029 and TJ-041 treated infected cells, only small sized fused cells with some nuclei were found. TJ-019 and TJ-041 slightly disrupted actin microfilaments, and TJ-001 and TJ-002 destroyed them. TJ-041 slightly disrupted microtubules, and TJ-001 and TJ-002 disrupted them. In general, the medicines effective on common cold and bronchitis inhibited hPIV-2 replication.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Ribavirin inhibits human parainfluenza virus type 2 replication in vitro.

The antiviral activities of eight nucleoside analog antiviral drugs (ribavirin, acyclovir, lamivudine, 3'-azido-3'-deoxythymidine, emtricitabine, tenofovir, penciclovir and ganciclovir) against human parainfluenza virus type 2 (hPIV-2) were investigated. Only ribavirin (RBV) inhibited both cell fusion and hemadsorption induced by hPIV-2. RBV considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV, as determined by real time PCR. An indirect immunofluorescence study showed that RBV largely inhibited viral protein synthesis. mRNAs of the proteins were not detected, indicating that inhibition of protein synthesis was caused by transcription inhibition by RBV. Using a recombinant green fluorescence protein-expressing hPIV-2 without matrix protein, it was found that RBV did not completely inhibit virus entry into the cells; however, it almost completely blocked multinucleated giant cell formation. RBV did not disrupt actin microfilaments and microtubules. These results indicate that the inhibitory effect of RBV is caused by inhibition of both virus genome and mRNA synthesis, resulting in inhibition of virus protein synthesis, viral replication and multinucleated giant cell formation (extensive cell-to-cell spreading of the virus).

Legume lectins inhibit human parainfluenza virus type 2 infection by interfering with the entry.

Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences.

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.

Fucoidan inhibits parainfluenza virus type 2 infection to LLCMK2 cells.

The effects of fucoidan and L-fucose, a fundamental major component of fucoidan, on the growth of human parainfluenza virus type 2 (hPIV-2) in LLCMK(2) cells were investigated. Fucoidan inhibited cell fusion and hemadsorption, but L-fucose only partly inhibited both. Virus RNA was not detected in the hPIV-2 infected cells cultured with fucoidan. However, L-fucose did not inhibit virus RNA synthesis. Indirect immunofluorescence study showed that virus protein synthesis was inhibited by fucoidan, but not by L-fucose. Furthermore, using a recombinant, green fluorescence protein-expressing hPIV-2, it was found that virus entry was inhibited by fucoidan, but not by L-fucose. These results suggested that fucoidan inhibited virus adsorption to the surface of the cells by binding to the cell surface and prevented infection, indicating that the sulfated polysaccharide form was important for the inhibition by fucoidan.

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system.

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.

Both type-I hemidesmosomes and adherens-type junctions contribute to the cell-substratum adhesion system in myoepithelial cells.

Myoepithelial cells present in exocrine glands cause secretion from the glands by contraction. They have mixed characteristics with regard to cytoskeletal elements, containing both epithelial-type intermediate filaments and smooth muscle-type myofilaments. For further characterization, myoepithelial cells from bovine apocrine sweat glands and tracheal glands were here examined with special attention to the cell-substratum adhesion system. Immunofluorescence microscopy using a panel of antibodies against adherens-type junctional and hemidesmosomal proteins demonstrated two types of cell-substratum junctions in myoepithelial cells from both glands. Type-I hemidesmosomes (HDs) consisting of plectin, BP230, integrin alpha6beta4, and BP180 were thus observed as punctate arrays longitudinally arranged along myoepithelial cell surfaces, while adherens-type junctions were similarly evident as linear rib-like structures. Double-label immunofluoresence revealed the two junctions to be distributed in a mutually exclusive or independent manner. Electron microscopy further demonstrated that apocrine myoepithelial cells surround secretory epithelial cells completely, without any gaps, HDs being abundant along the basement membrane, but with no distinct structures in the inter-hemidesmosomal regions. Immunoelectron microscopy, however, revealed an interhemidesmosomal localization of vinculin, pointing to the existence of adherens-type junctions. Secretory epithelial cells in tracheal glands were found not to be completely covered with myoepithelial cells, so that more than half of them are directly attached to the basement membrane, where they form type II-HDs lacking BP230 and BP180, but no detectable adherens junctions, like epidermal basal cells and sebaceous gland cells. These observations demonstrate that, in addition to their cytoskeleton, myoepithelial cells have both epithelial- and smooth muscle-type cell-substratum adhesion structures, i.e. HDs and dense plaque-like adherens junctions.

Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro: (mouse submandibular gland/branching morphogenesis/heparan sulfate proteoglycan/heparitinase).

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum. Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non-or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12-day gland inhibited the branch-initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [3 H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of 35 S-inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate. The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml-1 ) or heparin (75 µg ml-1 ), but not by chondroitin sulfate (1 mg ml-1 ) in the culture medium. Transmission electron microscopic study indicated that at the epithelial-mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase-treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.