Pathogenesis, transmissibility, and ocular tropism of a highly pathogenic avian influenza A (H7N3) virus associated with human conjunctivitis

Abstract

H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus (A/Mexico/InDRE7218/2012 [MX/7218]) and related avian H7 subtype viruses in mouse and ferret models. Several high- and low-pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tracts of ferrets; however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naive ferrets in a direct-contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet it downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naive-contact ferrets, while concurrently retaining the ability to replicate within ocular tissue and allowing the eye to serve as a portal of entry.

Fig 1

Comparison of morbidity and viral replication following influenza A H7 influenza virus infection in mice. BALB/c mice were inoculated i.n. with 10⁶ EID₅₀/50 μl of each virus indicated. (A) Weight loss was monitored for 14 days following inoculation (5 to 7 mice/group). Weight loss in MX/7218-infected mice is representative of that for 6/7 mice which succumbed to infection at this challenge dose. (B to H) Nose (white bars) and lung (black bars) tissues were collected on days 3 and 6 p.i. with HPAI H7N3 viruses (B and C), LPAI H7N3 viruses (D to F), and LPAI H7N9 viruses (G and H). Tissue homogenates (3 mice/group) were titrated in eggs, and the results are expressed as the mean log₁₀ EID₅₀/ml plus standard deviation. The limit of virus detection was 10^1.5 EID₅₀/ml.

Fig 2

Transmissibility of H7N3 and H7N9 influenza viruses after direct contact. Three ferrets were inoculated with 10⁶ EID₅₀ of the H7N3 virus MX/7218 or the H7N9 virus gs/NE/11, and nasal washes were collected from each ferret on the indicated days p.i. (left set of bars) to assess viral replication. A naive ferret was placed in the same cage as each inoculated ferret at 24 h p.i., and nasal washes were collected from each contact ferret on the indicated days postcontact (right set of bars). The limit of virus detection was 10^1.5 EID₅₀/ml.

Fig 3

Replication kinetics of H7N3 and H7N9 viruses in human respiratory cells. Calu-3 cells were infected with the indicated viruses at an MOI of 0.01. Supernatants were removed at the indicated times p.i., and titers of infectious virus were determined by standard plaque assay. The limit of virus detection was 10 PFU/ml. The mean from duplicate independent cultures per virus plus standard deviation is shown.

Fig 4

Fold regulation of selected genes related to NF-κB signaling in human respiratory cells following HPAI H7N3 virus infection. Calu-3 cells were infected with MX/7218 virus at an MOI of 2. Total RNA was isolated in triplicate from cells at 24 h p.i. and examined by real-time RT-PCR array analysis. Genes which exhibited a significant (P < 0.05) >3-fold change in expression over/under that in mock-infected cells are shown. Gray bars signify genes whose regulation is specific for the H7 subtype rather than other influenza viruses in this cell type (17).