Isolation of Primary Porcine Bronchial Epithelial Cells for Nipah Virus Infections.

The Malaysian strain of Nipah virus (NiV) first emerged in 1998/99 and caused a major disease outbreak in pigs and humans. While humans developed fatal encephalitis due to a prominent infection of brain microvessels, NiV-infected pigs mostly suffered from an acute respiratory disease and efficiently spread the infection via airway secretions. To elucidate the molecular basis of the highly productive NiV replication in porcine airways in vitro, physiologically relevant cell models that have maintained functional characteristics of airway epithelia in vivo are needed. Here, we describe in detail the method of isolating bronchial epithelial cells (PBEpC) from pig lungs that can be used for NiV infection studies. After the dissection of primary bronchia and removal of the mucus and protease digestion, bronchi segments are cut open and epithelial cells are scraped off and seeded on collagen-coated cell culture flasks. With this method, it is possible to isolate about 2 × 106 primary cells from the primary bronchi of one pig lung which can be cryopreserved or further subcultured. PBEpC form polarized monolayers on Transwell membrane inserts as controlled by immunostainings of epithelial marker proteins. NiV infection causes rapid formation of syncytia, allowing productive NiV infections in living PBEpC cultures to be monitored by phase-contrast microscopy.

Cytokine Induction in Nipah Virus-Infected Primary Human and Porcine Bronchial Epithelial Cells.

During the Nipah virus (NiV) outbreak in Malaysia, pigs and humans were infected. While pigs generally developed severe respiratory disease due to effective virus replication and associated inflammation processes in porcine airways, respiratory symptoms in humans were rare and less severe. To elucidate the reasons for the species-specific differences in NiV airway infections, we compared the cytokine responses as a first reaction to NiV in primary porcine and human bronchial epithelial cells (PBEpC and HBEpC, respectively). In both cell types, NiV infection resulted in the expression of type III interferons (IFN-λ). Upon infection with similar virus doses, viral RNA load and IFN expression were substantially higher in HBEpC. Even if PBEpC expressed the same viral RNA amounts as NiV-infected HBEpC, the porcine cells showed reduced IFN- and IFN-dependent antiviral gene expression. Despite this inherently limited IFN response, the expression of proinflammatory cytokines (IL-6, IL-8) in NiV-infected PBEpC was not decreased. The downregulation of antiviral activity in the presence of a functional proinflammatory cytokine response might be one of the species-specific factors contributing to efficient virus replication and acute inflammation in the lungs of pigs infected with the Malaysian NiV strain.

Variability of interferon-λ induction and antiviral activity in Nipah virus infected differentiated human bronchial epithelial cells of two human donors.

Highly pathogenic Nipah virus (NiV) generally causes severe encephalitis in humans. Respiratory symptoms are infrequently observed, likely reflecting variations in infection kinetics in human airways. Supporting this idea, we recently identified individual differences in NiV replication kinetics in cultured airway epithelia from different human donors. As type III interferons (IFN-λ) represent major players in the defence mechanism against viral infection of the respiratory mucosa, we studied IFN-λ induction and antiviral activity in NiV-infected primary differentiated human bronchial epithelial cells (HBEpCs) cultured under air-liquid interface conditions. Our studies revealed that IFN-λ was upregulated in airway epithelia upon NiV infection. We also show that IFN-λ pretreatment efficiently inhibited NiV replication. Interestingly, the antiviral activity of IFN-λ varied in HBEpCs from two different donors. Increased sensitivity to IFN-λ was associated with higher expression levels of IFN-λ receptors, enhanced phosphorylation of STAT1, as well as enhanced induction of interferon-stimulated gene expression. These findings suggest that individual variations in IFN-λ receptor expression affecting IFN responsiveness can play a functional role for NiV replication kinetics in human respiratory epithelial cells of different donors.

Species-specific and individual differences in Nipah virus replication in porcine and human airway epithelial cells.

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.