Ocular infection of mice with influenza A (H7) viruses: a site of primary replication and spread to the respiratory tract - PubMed (original) (raw)
Ocular infection of mice with influenza A (H7) viruses: a site of primary replication and spread to the respiratory tract
Jessica A Belser et al. J Virol. 2009 Jul.
Abstract
Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.
Figures
FIG. 1.
Morbidity and mortality following ocular inoculation with avian influenza viruses. BALB/c mice were inoculated by the ocular route with 106 EID50/5 μl of H7N7 (A and B) or H5N1 (C and D) viruses as indicated. Five to ten mice per virus were observed for weight loss (A and C) and survival (B and D) for 14 days p.i. Data are expressed as a percentage of mean starting weight or survival.
FIG. 2.
Comparison of mean titers of influenza viruses recovered from mouse tissues following ocular inoculation. BALB/c mice were inoculated by the ocular route with 106 EID50 or PFU/5 μl of Eurasian lineage H7N7 (A), North American lineage H7N3 and H7N2 (B), H5N1 (C), or H3N2 and H1N1 (D) viruses as indicated. Tissues were collected on days 3 and 6 p.i. from 3 to 6 mice per group. Tissue homogenates were prepared and titers were determined in eggs. Virus end point titers are expressed as mean log10 EID50/ml ± the standard error of the mean. The limit of virus detection was 100.8 EID50/ml (horizontal line).
FIG. 3.
Expression of α2-3- and α2-6-linked SAs on mouse and human ocular cells. Mouse corneal epithelial sheets (A) or HCEpiCs (B) were fixed and stained for α2-3 (MAAI and MAAII) and α2-6 (SNA) sialyl linkages as shown in green. The cells were counterstained with DAPI (blue) to detect cell nuclei. Neuraminidase treatment abolished staining to sialyl linkages (B insets).
FIG. 4.
Attachment of influenza viruses to mouse corneal and lung tissue. Mouse corneal (A) or lung (B) tissue was incubated with FITC-labeled virus overnight, treated with AEC, and counterstained with hematoxylin. FITC-labeled virus attachment representative of dose-dependent direct binding is shown in red. Neuraminidase-treated tissues are shown as insets in panel A. Tissues are shown at a magnification of ×400, with the exception of SC18 lung, shown at ×100.
FIG. 5.
Replication kinetics of influenza viruses in mouse ocular tissue. Whole corneas (A) or excised corneal epithelial sheets (B) were collected from naïve mice and infected ex vivo with 5 × 103 PFU/ml of each virus indicated. Culture supernatants were taken at the indicated times p.i., and titers were determined for infectious virus. The mean of five tissues per virus ± standard deviation is shown.
FIG. 6.
Replication kinetics of influenza viruses in primary HCEpiCs. HCEpiCs were infected at an MOI of 0.01 with the indicated viruses. Culture supernatants were taken at indicated times p.i., and titers were determined for infectious virus. The mean from duplicate independent cultures per virus ± standard deviation is shown.
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