UBE2NL protein in virus-infected or mock-infected MDCK cells at 6 h p.i..

<p>MDCK cells were infected with the viruses at MOI of 0.1 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were incubated for 6 h at 37°C in the maintenance media. Then, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to UBE2NL protein and NP protein. (A) The fluorescence images (10×) of the infected and mock-infected cells at 6 h p.i. The FITC-fluorescence signal was expressed as UBE2NL protein and TRITC-fluorescence signal was expressed as the infected cells. (B) The fluorescence images (60×) of the cells infected by rPR8-H9N2NA or rPR8-H5N1NA viruses.</p

Western blots of representative proteins in influenza virus-infected MDCKs.

<p>The samples were prepared from MDCK cells that were virus-infected or mock-infected cells at 6 h p.i.. The β-actin protein was used as a control. (A) Western blot confirmation of differentially expressed proteins for PSMC2 (C08) and UBE2NL (C26). (B) ImageJ software analysis of the ratios of proteins changes according to Fig. 4A.</p

List of differentially expressed protein spots in influenza virus-infected and mock-infected MDCKs identified by MALDI-TOF/TOF.

a<p>The arrow“↑” represents the identified proteins were upregulated and the arrow “↓”represents the identified proteins were downregulated.</p

Table_1_Rapid and Specific Detection of All Known Nipah virus Strains’ Sequences With Reverse Transcription-Loop-Mediated Isothermal Amplification.XLSX

Nipah virus (NiV) is a zoonotic virus and can be transmitted through contaminated food or directly between people. NiV is classified as a Biosafety Level 4 agent, not only because of its relatively high case fatality rate, but also because there is no vaccine or other medical countermeasures and it appears to be transmitted by fomites/particulates. The development of rapid detection assay for NiV is of great importance because no effective field test is currently available. In this study, an isothermal (65°C) reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method was developed, targeting the nucleocapsid protein (N) gene, for the rapid detection of NiV, and was compared with conventional RT-PCR. Three pseudoviruses of NiV N gene representing all known strains were constructed to replace live NiV. A set of RT-LAMP primers, targeting a highly conserved region of the N gene in the viral genome was designed to identify all known NiV strains. Sensitivity tests indicated that the detection limit of the RT-LAMP assay was approximately 100 pg of total NiV pseudovirus RNA, which is at least 10-fold higher than that of conventional RT-PCR. Specificity tests showed that there was no cross-reactivity with nucleocapsid protein gene of Hendra virus, Newcastle disease virus, Japanese encephalitis virus, or Influenza A virus. The RT-LAMP assay provides results within 45 min, and requires no sophisticated instruments, except an isothermal water bath or metal bath with 1 μl calcein indicator. An analysis of the clinical samples showed that the assay had good stability. In conclusion, systematic experiments have shown that the RT-LAMP assay developed here effectively detects three NiV pseudoviruses representing all known strains of NiV, with high specificity, sensitivity and stability.</p

Characterization of recombinant influenza viruses.

<p>(A) Multiple-cycle growth curves of recombinant viruses on MDCK cells. MDCK cells were infected at a multiplicity of infection (MOI) of 0.001. The virus titers were measured by TCID₅₀. The data shown are means ± SD for triplicate wells at each time point. Statistical significance (<i>p</i>>0.05). (B) SDS-PAGE analysis of expression of NA proteins in recombinant viruses. Concentrated viruses were resuspended with SDS-PAGE sample loading buffer, incubated at 37°C for 30 min, and heated at 100°C for 1 min. The protein was separated by 12% SDS-PAGE, gels were stained with coomassie brilliant blue G250.</p

Influenza virus induced cell-cell fusion. MDCK cells were infected with the viruses at MOI of 0.1 or 0.001 in the presence of 1 µg/ml TPCK-trypsin.

<p>After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated for the indicated times at 37°C in the maintenance media. At the indicated time, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to whole viruses. (A) MOI at 0.1, 3 h p.i. (B) MOI at 0.1, 6 h p.i. (C) MOI at 0.001, 12 h p.i.</p

Virus elution in <i>vitro</i>.

<p>50 µl two-fold dilutions of virus containing the HA titers of 1∶128 was incubated with 50 µl 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. Then microtiter plates were incubated at 37°C, and the reduction of HA titers was measured periodically for 8 h.</p

Protein expression profiles of the influenza- and mock-infected MDCK cells.

<p>Cell lysates (120 µg) were separated on 13-cm (isoelectric point [pI] 4–7) linear gradient IPG strips using 12.5% SDS-PAGE. Differentially expressed protein spots are indicated with green squares. (A) Representative 2-DE gels of influenza- and mock-infected MDCK cells. T1/C: PR8-wt infected/mock infected, T3/C: rH1N1NA infected/mock infected, T4/C: rH9N2NA infected/Mock infected, T5/C: rH5N1NA infected/mock infected, T3/T1: rH1N1NA infected/PR8-wt infected, T4/T1: rH9N2NA infected/PR8-wt infected, T5/T1: rH5N1NA infected/PR8-wt infected. (B) Numbers of differentially expressed protein spots detected by 2-DE in virus-infected MDCK cells compared with mock-infected MDCK cells. The number of spots ≥0 indicated the proteins were upregulated, and the number <0 indicated the proteins were downregulated. (C) Numbers of differentially expressed protein spots detected by 2-DE in recombinant viruses compared with wild-type virus (wt-PR8)-infected MDCK cells.</p

Impact of different NA on initiation of influenza virus infection.

<p>MDCK cells were infected at an MOI of 0.001 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated at 37°C for 6 h. At the indicated time, the cells were processed for immunofluorescence, and the infected cells were detected with polyclonal antisera to whole viruses. (A) Fluorescence images of the infected cells at 6 h p.i. Fluorescent photomicrographs showing the intracellular expression of virus protein in cell culture. The FITC-fluorescence signal was expressed as the infected cells. (B) Volocity Demo software analysis of the ratios of infected cells according to the Fig. 5A. *, statistical significance (<i>p</i><0.05) (C) Flow-cytometric analysis of virus-infected cells at 6 h p.i. MDCK cells (2×10⁶) in suspension were incubated with PBS or anti-PR8 antibodies on ice. Then the FITC-conjugated IgG secondary antibodies were added. After washing, the cells were fixed and the number of infected cells was determined by flow cytometric analysis. *, statistical significance (<i>p</i><0.05).</p

Transcriptional profiles of differentially expressed proteins in influenza virus-infected MDCKs.

<p>Total cellular RNA from MDCKs with or without influenza virus infection was subjected to real-time RT-PCR. Samples were normalized to mock-infected MDCKs using β-actin as the reference gene.</p