Oxygen-dependent changes in lung development do not affect epithelial infection with influenza A virus.

Infants born prematurely often require supplemental oxygen, which contributes to aberrant lung development and increased pulmonary morbidity following a respiratory viral infection. We have been using a mouse model to understand how early-life hyperoxia affects the adult lung response to influenza A virus (IAV) infection. Prior studies showed how neonatal hyperoxia (100% oxygen) increased sensitivity of adult mice to infection with IAV [IAV (A/Hong Kong/X31) H3N2] as defined by persistent inflammation, pulmonary fibrosis, and mortality. Since neonatal hyperoxia alters lung structure, we used a novel fluorescence-expressing reporter strain of H1N1 IAV [A/Puerto Rico/8/34 mCherry (PR8-mCherry)] to evaluate whether it also altered early infection of the respiratory epithelium. Like Hong Kong/X31, neonatal hyperoxia increased morbidity and mortality of adult mice infected with PR8-mCherry. Whole lung imaging and histology suggested a modest increase in mCherry expression in adult mice exposed to neonatal hyperoxia compared with room air-exposed animals. However, this did not reflect an increase in airway or alveolar epithelial infection when mCherry-positive cells were identified and quantified by flow cytometry. Instead, a modest increase in the number of CD45-positive macrophages expressing mCherry was detected. While neonatal hyperoxia does not alter early epithelial infection with IAV, it may increase the activity of macrophages toward infected cells, thereby enhancing early epithelial injury.

Generation and Characterization of Single-Cycle Infectious Canine Influenza A Virus (sciCIV) and Its Use as Vaccine Platform.

Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine.

Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA).

Avian-derived influenza A zoonoses are closely monitored and may be an indication of virus strains with pandemic potential. Both successful vaccination and convalescence of influenza A virus in humans typically results in the induction of antibodies that can neutralize viral infection. To improve long-standing and new-generation methodologies for detection of neutralizing antibodies, we have employed a novel reporter-based approach that allows for multiple antigenic testing within a single sample. Central to this approach is a single-cycle infectious influenza A virus (sciIAV), where a functional hemagglutinin (HA) gene was changed to encode either the green or the monomeric red fluorescent protein (GFP and mRFP, respectively) and HA is complemented in trans by stable HA-expressing cell lines. By using fluorescent proteins with non-overlapping emission spectra, this novel bivalent fluorescence-based microneutralization assay (BiFMA) can be used to detect neutralizing antibodies against two distinct influenza isolates in a single reaction, doubling the speed of experimentation while halving the amount of sera required. Moreover, this approach can be used for the rapid identification of influenza broadly neutralizing antibodies. Importantly, this novel BiFMA can be used for any given influenza HA-pseudotyped virus under BSL-2 facilities, including highly pathogenic influenza HA isolates.