In Vitro Investigation of the Interaction of Avian Metapneumovirus and Newcastle Disease Virus with Turkey Respiratory and Reproductive Tissue

Abstract

In poultry, several respiratory viral infections lead to a drop in egg production associated with high economic losses. While the virus-host interactions at the respiratory epithelium are well studied, less is known about these interactions in the oviduct. To investigate possible differences between virus infections at these epithelial structures, we compared the interactions of two important poultry viruses on turkey organ cultures. Two members of the order Mononegavirales, the Avian Metapneumovirus (AMPV) and the Newcastle disease virus (NDV), were selected to conduct the in vitro experiments since these viruses can infect both the trachea and oviduct. In addition, we used different strains of these viruses, a subtype A and a subtype B strain for AMPV and the NDV strains Komarow and Herts'33, to detect possible differences not only between the tissues but also between different viral strains. Turkey tracheal and oviduct organ cultures (TOC and OOC) were prepared to investigate viral replication, antigen localisation, lesion development, and the expression pattern of interferon-λ and importin-α isoforms. All viruses replicated more efficiently in the oviduct than in the tracheal epithelium (p < 0.05). In addition, we observed higher expression levels of both, IFN-λ and importin-α in OOCs compared to TOCs. Our results indicated strain-dependent differences, with the AMPV-B- and Herts'33 strains being more virulent in organ cultures than the AMPV-A- and Komarow strains, based on the higher viral genome loads, more severe histological lesions, and higher upregulation of IFN-λ. Overall, our findings reveal tissue- and virus strain-dependent differences, which may have consequences for disease development in the host tissue and, subsequently, possible treatment strategies.

Keywords: avian metapneumovirus; importin-alpha; newcastle disease virus; oviduct organ culture (OOC); tracheal organ culture (TOC); virus–host interactions.

Figure 1

Comparative quantification of viral genome using qRT-PCR in TOCs and OOCs after inoculation with AMPV-A (A), AMPV-B (B), NDV-HS (C), or NDV-KO (D). For each group, three to five rings were collected at 1, 24, 48, 72, and 96 hpi and processed for viral quantification. Normalised data are presented as mean 40 - Ct. Error bars represent standard deviations (SD). * indicates significant differences between different inoculation time points for each virus, determined using the Tukey HSD all-pairwise comparison test. Small letters indicate significant differences between TOCs and OOCs at the same time point for each virus, determined using two-sample t-tests. p-value < 0.05. Graphs represent data from one representative experiment.

Figure 2

Comparison of AMPV-B viral genome in TOCs and OOCs between two experiment series. For each group, three to five rings were collected at 1, 24, 48, 72, and 96 hpi and processed for AMPV-B quantification in TOCs (A) and OOCs (B) using qRT-PCR. Normalised data are presented as mean 40 - Ct. Error bars represent standard deviations (SD). Small letters indicate significant differences between the two experiments for either the trachea or oviduct at the same time point (A,B), determined using two-sample t-tests. p-value < 0.05.

Figure 3

Antigen detection of AMPV-A (A,C) and NDV-HS (B,D) in TOCs (A,B) and OOCs (C,D) through immunohistochemistry. Brown staining indicates virus-infected cells. (A) TOC infected with AMPV-A, (B) TOC infected with NDV-HS, (C) OOC infected with AMPV-A, (D) OOC infected with NDV-HS, (E) virus-free TOC, (F) and virus-free OOC—all at 48 hpi. Arrowheads mark specific viral antigen staining, and black boxes visualize zoomed-in areas (shown in B.2,C.2). The scale bar was 50 µm.

Figure 4

Ciliostasis in TOCs after inoculation with either AMPV-A, AMPV-B, NDV-KO, or NDV-HS. The daily average ciliary activity for ten TOCs per group is displayed. Error bars indicate standard deviations (SD). Small letters represent significant differences between the four different virus strains and the negative control at the same time points, at p < 0.05, using the Kruskal–Wallis all-pairwise comparison test. Graphs represent data from one representative experiment.

Figure 5

Histopathological lesion development at the epithelial surface of TOCs (A,B) and OOCs (C,D). Images are AMPV-B-infected TOCs at (A) 24 hpi and (B) 72 hpi, and NDV-HS-infected OOCs at (C) 24 hpi and (D) 96 hpi. (E) TOC and (F) OOC illustrate virus-free rings at time point 96 hpi. Arrowheads highlight cilia loss. The scale bar is 50 µm.

Figure 6

Quantification of IFN-λ mRNA expression in correlation with AMPV and NDV replication in TOCs. (A) AMPV-A, (B) AMPV-B, (C) NDV-HS, and (D) NDV-KO. Five rings/group/time points were investigated. Error bars indicate standard deviations. * indicate significant differences between the virus-inoculated group and the virus-free group at the same time point, determined using the Tukey HSD all-pairwise comparison test (ANOVA, p < 0.05). The graphs show data from a single representative experiment.

Figure 7

Quantification of IFN-λ mRNA expression in correlation with AMPV and NDV replication in OOCs. (A) AMPV-A, (B) AMPV-B, (C) NDV-HS, and (D) NDV-KO. Three rings/group/time points were investigated. Error bars indicate standard deviations. * indicate significant differences between the virus-inoculated group and the virus-free group at the same time point, determined using the Tukey HSD all-pairwise comparison test (ANOVA, p < 0.05). The graphs depict data from one representative experiment.

Figure 8

Quantification of mRNA expression levels of importin-α isoforms in TOCs and OOCs after infection with AMPV or NDV. The different mRNA expression patterns of importin-α 1, 3, 4, 5, and 7 were detected after the infection of TOCs (A,C,E,G) and OOCs (B,D,F,H) with either (A,B) AMPV-A, (C,D) AMPV-B, (E,F) NDV-HS, or (G,H) NDV-KO. * indicate significant differences between the virus-inoculated group and the virus-free group at the same time point, determined using the Tukey HSD all-pairwise comparison test (ANOVA p < 0.05). The graphs represent data from one representative experiment.