Cell lines and viruses
Vero cells were maintained in Dulbecco's modified Eagle’s medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, DMEM-10FBS). The NiV strain, Ma-JMR-01-98, was kindly provided by Dr. Kouichi Morita (Institute for Tropical Medicine, Nagasaki University, Nagasaki, Japan). The virus was propagated in Vero cells. At 2 days post-infection, the infectious fluid was harvested. Cellular debris was removed by low-speed centrifugation (2,000×g, 10 min, 4°C) and the supernatant was collected. Experiments using infectious NiV were conducted in BSL-3 or BSL-4 laboratories under the biosafety regulation of the National Institute of Infectious Diseases, Tokyo, Japan.
Heat treatment and UV irradiation
The NiV strain Ma-JMR-01-98 (3.2 × 107 50% tissue culture infectious dose (TCID50)/mL) was diluted 10-fold in DMEM-10FBS or in pooled human serum (Tennessee Blood Services, Memphis, TN) to prepare virus samples (3.2 × 106 TCID50/mL) for the inactivation experiments. Next, 200 µL of the virus sample was aliquoted into a 1.5 mL transparent polypropylene tube (No.72.692S, Sarstedt, Nümbrecht, Germany). The virus samples were then processed by heating at 56°C or 60°C for 30 or 60 min or by UV irradiation (wavelength, 312 nm; output power, 2.5 mW/cm2) for 10 min or 30 min using a transilluminator (model TPP-10M, Vilber-Lourmat, Collégien, France). The distance between the UV outlet and samples was approximately 1.0 cm and the UV intensity was 860 μW/cm2. To ensure the delivery of UV irradiation onto the whole surface of the sample tubes, tubes on the transilluminator were covered with aluminum foil, which reflects UV effectively , as shown in the schematic overview of the inactivation method (Fig. 1). All samples were stored at -80°C until virus titration.
The infectious dose of NiV in the samples with or without heating and/or UV-treated samples was determined as described previously . Briefly, all samples were serially 10-fold diluted with DMEM, and 33 μL volumes of each dilution were placed onto the confluent monolayers of Vero cells cultured in 96-well microplates. Following a 3-day incubation at 37°C in 5% CO2, the plates were scored for cytopathic effect (CPE), and the TCID50 was determined.
Confirmation of complete inactivation of spiked NiV by virus isolation
Two hundred microliters of DMEM-10FBS or pooled human serum spiked with infectious NiV (6.4 ×105 TCID50) was prepared as described above. The sample-containing tubes with aluminum foil lids were then treated with UV irradiation for 30 min. Subsequently, the UV-treated tubes were further heated at 56°C for 30 min in a water bath. The samples with the spiked virus, which were not subjected to the treatment, were used as controls. Triplicate samples with the treatment and the controls were tested for virus isolation using Vero cells. Vero cells seeded in a 6-well plate were inoculated with 100 μL of each sample. CPE in the cells was monitored for 6 days, following which the plates were stored at -80 °C. After thawing the plates, the supernatant of the culture fluid was collected by low-speed centrifugation (2000×g, 5 min), and 900 μL of the supernatant was transferred onto Vero cells cultured in a new plate. After 1 h incubation, the supernatant was replaced with fresh growth medium (DMEM-10FBS), and the cells were incubated for 6 or 7 days to observe the CPEs. This blind passage was repeated three times.
Stability of NiV spiked in pooled serum at room temperature
The stock of NiV containing 3.2 × 107 TCID50/mL was diluted 10-fold in the pooled human serum to prepare the virus samples (3.2 × 106 TCID50/mL). Two hundred microliters of the virus sample in the transparent polypropylene tube (No.72.692S, Sarstedt) was then incubated at 25°C for 7 days, followed by storage at -80°C. In contrast, the virus sample was desiccated by spreading 20 µL onto 6-well plastic plates and air drying at 25°C within 7 days. The virus was recovered at intervals by resuspension in 200 µL of DMEM-10FBS and stored at -80°C. The infectious doses of all the samples stored were determined in Vero cells as described above.