Quantitative estimation of Nipah virus replication kinetics in vitro

Abstract

Background: Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay.

Results: The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/microL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every approximately 40 minutes and attained peak intracellular virus RNA level of approximately 8.4 log PFU/microL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at approximately 7.9 log PFU/microL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was approximately 0.07 log PFU/muL per hour and less than 10% of the released Nipah virus RNA was infectious.

Conclusion: The SYBR Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection.

Figure 1

Changes in Vero cell morphology following Nipah virus infection. Cell fusion and syncytial formation were observed at eight hours PI (b, thick arrow). Multinucleated giant cells were noted to increase in frequency at 32 hours PI (c, thick arrow). Evidence of apoptosis with the presence of blebbing cell and apoptotic bodies was noted at 48 hours PI (d, thin arrow). At 64 hours PI onwards, cells started to detach from the surface of the tissue culture flask (e). The inset in (c) is an electron micrograph showing multinucleated cells (N) at 32 hours PI and the presence of nuclear invagination (thin arrowhead). The mock-infected Vero cells at 72 hours PI is shown in (f).

Figure 2

Sensitivity and specificity of one-tube qRT-PCR for detection of Nipah virus RNA. DNA fragments obtained from the RT-PCR were visualized in ethidium bromide-stained agarose gel (a). Input Nipah virus RNA in equivalent log PFU is indicated above the lanes. RNA extracted from mock-infected Vero cells and the Nipah virus Armored RNA^® served as the negative (neg) and positive (pos) controls, respectively. Lane (M) consisted of DNA molecular mass marker. Amplification plot of the SYBR^® Green I dye-based qRT-PCR assay were obtained from tenfold serial diluted Nipah virus RNA (1 × 10⁶ to 1 PFU) as indicated in (b). RNA extracted from mock-infected Vero cells was used as the negative control (NTC). The standard curve for the qRT-PCR (c) was generated using the same dilution series of Nipah virus RNA as the amplification plot. Correlation between log PFU/μL of infectious virus against total copy number of Nipah virus RNA (log RNA copy/μL) obtained from the qRT-PCR is shown in (d). Specificity of the assay was assessed and the difference in the melting temperature of the amplified DNA of Nipah virus (thick arrow) and Hendra virus (thin arrow) is indicated in the melting curve analysis (e).

Figure 3

Nipah virus replication in Vero cells. Vero cells were infected with Nipah virus at MOI of 0.2. At selected intervals, total RNA was isolated and the Nipah virus RNA levels were quantified using the SYBR^® Green I-based qRT-PCR assay in equivalent log PFU. A latent phase of at least eight hours followed by an exponential increase in the virus RNA level were noted for the intracellular Nipah virus RNA.