Role of interferon in the replication of human parainfluenza virus type 1 wild type and mutant viruses in human ciliated airway epithelium

Abstract

Human parainfluenza virus type 1 (HPIV1) is a significant cause of pediatric respiratory disease in the upper and lower airways. An in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, was used in this study to show that HPIV1 selectively infects ciliated cells within the HAE and that progeny virus is released from the apical surface with little apparent gross cytopathology. In HAE, type I interferon (IFN) is induced following infection with an HPIV1 mutant expressing defective C proteins with an F170S amino acid substitution, rHPIV1-C(F170S), but not following infection with wild-type HPIV1. IFN induction coincided with a 100- to 1,000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for the inhibition of the innate immune response. Two recently characterized live attenuated HPIV1 vaccine candidates expressing mutant C proteins were also evaluated in HAE. The vaccine candidates, rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) and rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11), which contain temperature-sensitive (ts) attenuating (att) and non-ts att mutations, were highly restricted in growth in HAE at permissive (32 degrees C) and restrictive (37 degrees C) temperatures. The viruses grew slightly better at 37 degrees C than at 32 degrees C, and rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) was less attenuated than rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11). The level of replication in HAE correlated with that previously observed for African green monkeys, suggesting that the HAE model has potential as a tool for the preclinical evaluation of HPIV1 vaccines, although how these in vitro data will correlate with vaccine virus replication in seronegative human subjects remains to be seen.

FIG. 1.

rHPIV1 wt infects HAE cells, spreads throughout the culture, and replicates efficiently. HAE cells were mock infected or infected with rHPIV1 wt at a low MOI (0.01 TCID₅₀/cell). At days 1 to 7 p.i., cells were fixed and stained en face for HPIV1 antigen (green) (A), and virus titers in the apical compartments were determined (B). Virus titers shown are the means of 3 to 11 cultures from a single donor ± standard errors (SE). The limit of detection is 1.2 log₁₀ TCID₅₀/ml, as indicated by the dashed line.

FIG. 2.

Comparison of single-cycle virus growth curves in HAE inoculated with rHPIV1 wt (A) or rHPIV1-C^F170S (B) at an MOI of 5.0 TCID₅₀/cell or with VSV (C) at an MOI of 4.2 PFU/cell at 37°C. Virus titers in the apical and basolateral compartments were determined at 8, 24, 48, and 72 h p.i. Virus titers shown are the means of cultures from two donors ± SE, and the limit of detection is 1.2 log₁₀ TCID₅₀/ml.

FIG. 3.

HPIV1 infection of ciliated cells without overt cytotoxicity. HAE were inoculated with HPIV1 wt or rHPIV1-C^F170S at an MOI of 5.0 TCID₅₀/cell or were mock infected, and cells were processed at 24 and 48 h p.i. for histological analysis in cross section by immunofluorescence (A) or hematoxylin and eosin staining (B) at ×40 magnification or stained en face (C). For histological immunofluorescence (A and C), antibodies to HPIV1 (green) and acetylated alpha-tubulin (red) were used to detect virus antigen and ciliated cells, respectively. Scale bars represent 20 μm (A and B) and 40 μm (C).

FIG. 4.

Comparison of the type I IFN response in HAE inoculated with rHPIV1 wt and rHPIV1-C^F170S. HAE were inoculated with rHPIV1s (MOI = 5.0 TCID₅₀/cell) or VSV (MOI = 4.2 PFU/cell) or were mock infected, and levels of type I IFN mRNA and secreted protein were quantitated at 8, 24, 48, and 72 h p.i. A type I IFN bioassay was used to quantitate levels of secreted type I IFN in the apical (A) and basolateral (B) compartments compared to those of an IFN-β standard. Type I IFN concentrations are expressed in pg/ml ± SE and are the means of data from duplicate cultures. The IFN-β standard has a specific activity of approximately 1 IU of antiviral activity per 5 pg. The limit of detection for type I IFN was 20.2 pg/ml. IFN-β mRNA expression was quantitated by qRT-PCR (C). Total RNA was extracted from HAE at 8, 24, 48, and 72 h p.i., and IFN-β mRNA was measured by qRT-PCR using specific primers and Taqman probes that were previously described (53). For each sample, the level of IFN-β mRNA was relative to that of β-actin and expressed as the increase compared to that for the mock-inoculated sample.

FIG. 5.

Virus replication and type I IFN production during multicycle growth curves in HAE inoculated with rHPIV1 wt and rHPIV1-C^F170S at an MOI of 0.01 TCID₅₀/cell at 37°C. Virus titers (log₁₀ TCID₅₀/ml) (line graph) and type I IFN concentrations (pg/ml) (bar graph) in apical washes were determined on each day from day 0 to day 7 p.i. The titers shown are means of data from duplicate donor cultures ± SE. The limit of detection for virus titers was 1.2 log₁₀ TCID₅₀/ml, and that for type I IFN was 31.1 pg/ml. The area shaded in gray represents the overall difference in virus replication between rHPIV1 wt and rHPIV1-C^F170S after day 2 p.i.

FIG. 6.

The ability of HPIV1 vaccine candidates to replicate in HAE at 32°C and 37°C was determined by multicycle growth curves. HAE were inoculated with rHPIV1 wt, rHPIV1-C^R84G/Δ170HN^T553AL^Y942A, or rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710-11 at an MOI of 0.01 TCID₅₀/cell at 32°C and 37°C. Virus titers (log₁₀ TCID₅₀/ml) in apical and basolateral compartments were determined each day from days 0 to 7 p.i. The virus titers shown are the means of data from triplicate donor cultures ± SE for apical washes (samples from the basolateral compartments were negative for virus), and the limit of detection is 1.2 log₁₀ TCID₅₀/ml.