Kinetics of viral replication and induction of host responses in ferrets differs between ocular and intranasal routes of inoculation

Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Virology (Impact Factor: 3.32). 02/2013; 438(2). DOI: 10.1016/j.virol.2013.01.012
Source: PubMed


While influenza viruses are typically considered respiratory pathogens, the ocular system represents a secondary entry point for virus to establish a productive respiratory infection and the location for rare instances of virus-induced conjunctivitis. We used the ferret model to conduct a side-by-side comparison of virus infectivity, kinetics of viral replication, and induction of host responses following inoculation by either the intranasal or ocular routes with two viruses, A/Netherlands/230/03 (H7N7) and A/Panama/2007/99 (H3N2). We show that ocular inoculation resulted in delayed virus replication and reduced levels of proinflammatory cytokine and chemokine transcript in respiratory tract but not ocular tissues compared with intranasally inoculated animals. We identified numerous proinflammatory mediators with known roles in ocular disease elicited in ferret eye tissue following influenza virus infection. These findings provide a greater understanding of the modulation of host responses following different inoculation routes and underscore the risk associated with ocular exposure to influenza viruses.

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    • "Expression of cytokine and chemokine genes has been assessed in ex vivo samples following infection of naïve or vaccinated ferrets with influenza virus or SARS-CoV by microarray analysis (Cameron et al., 2008; Fang et al., 2010; Rowe et al., 2010). Cytokine and chemokine gene profiles have also been assessed ex vivo and in in vitro epithelial cultures using SYBR green real time RT-PCR assays (Svitek and von Messling, 2007; Cameron et al., 2008; Danesh et al., 2008, 2011; Svitek et al., 2008; Kim et al., 2009; Fang et al., 2010; Hamelin et al., 2010; Kobinger et al., 2010; Rowe et al., 2010; Kang et al., 2011; Meunier and von Messling, 2011, 2012; Pillet et al., 2011; Huang et al., 2012; Maines et al., 2012; Meunier et al., 2012; Belser et al., 2013; Zeng et al., 2013). TaqMan chemistry incorporates target-specific fluorescent labeled probes enabling multiple genes can be assessed in a single real time PCR reaction (Giulietti et al., 2001). "
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    ABSTRACT: The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
    Journal of virological methods 05/2014; 205. DOI:10.1016/j.jviromet.2014.04.014 · 1.78 Impact Factor
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    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 tract of ferrets, however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naïve 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 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 naïve contact ferrets, while concurrently retaining the ability to replicate within ocular tissue allowing the eye to serve as a portal of entry.
    Journal of Virology 03/2013; 87(10). DOI:10.1128/JVI.00154-13 · 4.44 Impact Factor
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    ABSTRACT: Unlabelled: Respiratory pathogens have traditionally been studied by examining the exposure and infection of respiratory tract tissues. However, these studies typically overlook the role of ocular surfaces, which represent both a potential site of virus replication and a portal of entry for the establishment of a respiratory infection. To model transocular virus entry in a mammalian species, we established a novel inoculation method that delivers an aerosol inoculum exclusively to the ferret ocular surface. Using influenza virus as a representative respiratory pathogen, we found that both human and avian viruses mounted productive respiratory infections in ferrets following ocular-only aerosol inoculation, and we demonstrated that H5N1 virus can result in a fatal infection at doses below 10 PFU or with exposure times as short as 2 min. Ferrets inoculated by the ocular aerosol route with an avian (H7N7, H7N9) or human (H1N1, H3N2v) virus were capable of transmitting the virus to naïve animals in direct-contact or respiratory-droplet models, respectively. Our results reveal that ocular-only exposure to virus-containing aerosols constitutes a valid exposure route for a potentially fatal respiratory infection, even for viruses that do not demonstrate an ocular tropism, underscoring the public health implications of ocular exposure in clinical or occupational settings. Importance: In the absence of eye protection, the human ocular surface remains vulnerable to infection with aerosolized respiratory viruses. In this study, we present a way to inoculate laboratory mammals that excludes respiratory exposure, infecting ferrets only by ocular exposure to influenza virus-containing aerosols. This study demonstrates that the use of respiratory protection alone does not fully protect against influenza virus exposure, infection, and severe disease.
    Journal of Virology 06/2014; 88(17). DOI:10.1128/JVI.01067-14 · 4.44 Impact Factor

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