Amelioration of influenza virus pathogenesis in chickens attributed to the enhanced interferon-inducing capacity of a virus with a truncated NS1 gene

Abstract

Avian influenza virus (AIV) A/turkey/Oregon/71-SEPRL (TK/OR/71-SEPRL) (H7N3) encodes a full-length NS1 protein and is a weak inducer of interferon (IFN). A variant, TK/OR/71-delNS1 (H7N3), produces a truncated NS1 protein and is a strong inducer of IFN. These otherwise genetically related variants differ 20-fold in their capacities to induce IFN in primary chicken embryo cells but are similar in their sensitivities to the action of IFN. Furthermore, the weak IFN-inducing strain actively suppresses IFN induction in cells that are otherwise programmed to produce it. These phenotypic differences are attributed to the enhanced IFN-inducing capacity that characterizes type A influenza virus strains that produce defective NS1 protein. The pathogenesis of these two variants was evaluated in 1-day-old and 4-week-old chickens. The cell tropisms of both viruses were similar. However, the lesions in chickens produced by the weak IFN inducer were more severe and differed somewhat in character from those observed for the strong IFN inducer. Differences in lesions included the nature of inflammation, the rate of resolution of the infection, and the extent of viral replication and/or virus dissemination. The amelioration of pathogenesis is attributed to the higher levels of IFN produced by the variant encoding the truncated NS1 protein and the antiviral state subsequently induced by that IFN. The high titer of virus observed in kidney tissue ( approximately 10(9) 50% embryo lethal doses/g) from 1-day-old chickens infected intravenously by the weak IFN-inducing strain is attributed to the capacity of chicken kidney cells to activate the hemagglutinin fusion peptide along with their unresponsiveness to inducers of IFN as measured in vitro. Thus, the IFN-inducing capacity of AIV appears to be a significant factor in regulating the pathogenesis, virulence, and viral transmission of AIV in chickens. This suggests that the IFN-inducing and IFN induction suppression phenotypes of AIV should be considered when characterizing strains of influenza virus.

FIG. 1.

Schematic of NS1 proteins encoded by TK/OR/71-SEPRL and TK/OR/71-delNS1. (A) The full-length TK/OR/71-SEPRL NS1 protein consists of 230 amino acids. (B) The truncated NS1 protein from TK/OR/71-delNS1 consists of 124 amino acids. NLS, nuclear localization signal; NES, nuclear export signal; aa, amino acids; 38* represents the only amino acid required for RNA binding but not dimerization (42).

FIG. 2.

Experimental studies in 1-day-old and 4-week-old chickens inoculated with TK/OR/71-SEPRL or TK/OR/71-delNS1. (a, c, e, and g) Photomicrographs of hematoxylin- and eosin-stained tissue sections. (b, d, f, and h) Photomicrographs of tissue sections stained immunohistochemically to demonstrate avian influenza virus nucleoprotein. (a) Lymphocytic to heterophilic sinusitis with necrosis of respiratory epithelium in a 4-week-old chicken challenged with TK/OR/71-SEPRL via the intrachoanal cleft at 3 dpi. Bar, 25 μm. (b) Avian influenza nucleoprotein in respiratory epithelium of the infraorbital sinus in a 4-week-old chicken challenged with TK/OR/71-SEPRL via the intrachoanal cleft at 3 dpi. Bar, 10 μm. (c) Lymphocytic sinusitis with intact ciliated respiratory epithelium from a 4-week-old chicken challenged with TK/OR/71-delNS1 by intrachoanal cleft at 3 dpi. Bar, 25 μm. (d) Rare avian influenza nucleoprotein in respiratory epithelium of the infraorbital sinus from chicken in c. Bar, 10 μm. (e) Severe necrosis in kidney tubules from a 1-day-old chick intravenously challenged with TK/OR/71-SEPRL at 3 dpi. Bar, 50 μm. (f) Extensive staining of avian influenza nucleoprotein in necrotic kidney tubules from the chick in e. Bar, 25 μm. (g) Mild focal heterophilic interstitial nephritis with associated tubule necrosis in kidney from a 1-day-old chick intravenously challenged with TK/OR/71-delNS1 at 3 dpi. Bar, 50 μm. (h) Infrequent staining for avian influenza nucleoprotein in necrotic tubule epithelium and macrophages from the chick described in g. Bar, 25 μm.

FIG. 3.

Comparison of the IFN induction dose (multiplicity)-response (IFN yield) curves for TK/OR/71-delNS1 in monolayers of developmentally aged primary CEC and primary CEK cells from 18-day-old embryos. CEC were made from 10-day-old embryos and incubated in vitro for 9 days (total developmental age, 19 days) before inducing IFN. CEK cells were made from the kidneys of 18-day-old embryos and incubated in vitro for 2 days (total developmental age, 20 days) before inducing IFN. At the time of induction, there were 10⁷ cells per monolayer. The infected/induced cells were incubated at 40.5°C for 24 h, and the medium was harvested, processed, and assayed for IFN as described previously (47).

FIG. 4.

Comparison of the survival of TK/OR/71-SEPRL and TK/OR/71-delNS1 infectious virus measured by plaque formation on primary CEK cell monolayers as a function of ChIFN-α dose. Confluent monolayers of CEK cells prepared from 18-day-old embryos were treated for 24 h with various doses of rChIFN-α, infected with virus, incubated at 37.5°C for 3 days, and stained with neutral red; the number of plaques were determined relative to untreated cells; and the fraction of surviving PFP was determined. The triphasic slopes of the curves generated are considered experimentally indistinguishable and have been observed and analyzed previously with other AIV strains (45).