Microparticle and anti-influenza activity in human respiratory secretion.

Respiratory secretions, such as saliva and bronchoalveolar fluid, contain anti-influenza activity. Multiple soluble factors have been described that exert anti-influenza activity and are believed to be responsible for the anti-influenza activity in respiratory secretions. It was previously shown that a bronchial epithelial cell culture could produce exosome-like particles with anti-influenza activity. Whether such extracellular vesicles in respiratory secretions have anti-influenza activity is unknown. Therefore, we characterized bronchoalveolar lavage fluid and found microparticles, which mostly stained positive for epithelial cell markers and both α2,3- and α2,6-linked sialic acid. Microparticles were purified from bronchoalveolar lavage fluid and shown to exhibit anti-influenza activity by a hemagglutination inhibition (HI) assay and a neutralization (NT) assay. In addition, physical binding between influenza virions and microparticles was demonstrated by electron microscopy. These findings indicate that respiratory microparticles containing viral receptors can exert anti-viral activity by probably trapping viral particles. This innate mechanism may play an important role in the defense against respiratory viruses

Ultrastructural Features of Human Liver Specimens from Patients Who Died of Dengue Hemorrhagic Fever

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA–indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed

Surface markers of microparticle.

<p>BAL was double stained with annexin V and cell surface markers and analyzed by a flow cytometer: isotype control (A), CD11b (B), CD41a (C), CD45 (D), keratan sulfate (E), and SPD (F). MPs were gated using 1.33 μm beads as size markers, and the annexin V-positive gate was analyzed for the expression of the surface markers. To distinguish MPs from apoptotic bodies, BAL was double stained with annexin V and PI (G) using apoptotic bodies from hydrogen peroxide treated HT29 cells as a positive control (H). To detect surface sialic acid on MPs, BAL was double stained with annexin V and lectins. Annexin V-positive gate was analyzed for the expression of SNA (I) and MAA1 (J). The histrogram are representative of ten BAL samples.</p

Anti-viral activity by HI against H3N2 influenza A virus of BAL MP preparations with and without the sialidase treatment.

<p>(A) Pooled and purified BAL MP was mixed with PBS or RDE at a 1:3 ratio. The samples were rotated at 4°C overnight followed by sialidase inactivation at 56°C for 30 minutes. A sham treated sample was also tested in parallel. The data were derived from 6 pooled BAL samples run in duplicate. The HI titers are shown as the geometric mean±SEM and compared by a <i>t</i>-test. The three asterisks (***) represent a significant p-value < 0.0001 by ANOVA. (B) The sialidase treatment did not permanently inactivate the viral infectivity. A/Thailand/Siriraj-04/2003 (H3N2) virus was incubated with either MP or media at 37°C for 1 hour. RDE at a 1:2 ratio was added into either the virus only or the MP+virus and incubated at 4°C overnight. After the incubation, the virus was inoculated into MDCK cells and the viral NP protein was measured after an overnight incubation. The data represented a triplicate result for each condition. The two asterisks (**) represent a significant p-value < 0.01 by the <i>t</i>-test.</p

Transmission electron microscopy of the high-speed-centrifuged BAL pellet mixed with the viral preparation.

<p>(A) Binding of MP with the smooth surface (narrow arrow) and influenza virion with spikes (A/Thailand/104/2009) (wide arrow); (B) binding of MP (narrow arrow) with multiple virions (wide arrow); and (C) free exosome and MP with smooth membrane appearance shown with narrow and wide black arrows, respectively. The exosome size was usually smaller than 100 nm, whereas the MP was 100–1000 nm in diameter.</p

BAL fractionation using FPLC with a Hiprep sephacryl S-500 16/60 column and anti-viral activity in each BAL fraction.

<p>(A) The UV (280 nm) absorbance peak as an indicator of the protein concentration of the virus (A/Thailand/MVCU-013/2009) preparation. (B) The UV absorbance peaks of one BAL sample (upper) together with HI titers against pdmH1N1 (A/Thailand/104/2009) from 2 BAL samples (2 sets of fractions) (lower). The HI assay was run in duplicate and repeated by another set of BAL fractions. Each point of the HI titers is shown as the mean±SEM. The molecular mass at the peak of the HI titers was estimated from a dextran molecular weight standard.</p