Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)

Abstract

BackgroundThere is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a animal model for humans in influenza pathogenicity and transmissibility studies. In this manuscript, a nose-only bioaerosol inhalation exposure system that was recently developed and validated was used in an inhalation exposure study of aerosolized A/Vietnam/1203/2004 (H5N1) virus in ferrets. The clinical spectrum of influenza resulting from exposure to A/Vietnam/1203/2004 (H5N1) through intranasal verses inhalation routes was analyzed.ResultsFerrets were successfully infected through intranasal instillation or through inhalation of small particle aerosols with four different doses of Influenza virus A/Vietnam/1203/2004 (H5N1). The animals developed severe influenza encephalomyelitis following intranasal or inhalation exposure to 101, 102, 103, or 104 infectious virus particles per ferret.ConclusionsAerosolized Influenza virus A/Vietnam/1203/2004 (H5N1) is highly infectious and lethal in ferrets. Clinical signs appeared earlier in animals infected through inhalation of aerosolized virus compared to those infected through intranasal instillation.

Influenza virus aerosol exposure and analytical system for ferrets

“…The FID 50 of ARdelivered virus was 1.9 pfu for PN99 virus and 4 pfu for TH16 virus, similar to the reported 50% human infectious dose of aerosols 1-3 μm in size, which ranges from 0.6 to 3 50% tissue culture infectious dose (TCID 50 ) (20). A recently developed nose-only influenza aerosol system generates particles in the same size range as our system, but characterization of infectivity, transmission, and deposition in ferrets was not reported (21,22). Aerosol inoculation of mice and guinea pigs has been reported, but aerosol system validation and dosimetry were not described (23,24).…”

“…Ferrets, 6-to 11-mo-old (Triple F Farms) and serologically negative against currently circulating influenza viruses and H5N1 viruses, were used in this study and were housed in cages within a Duo-Flo Bioclean mobile clean room (Lab Products). Ferrets were sedated as described (10) (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) ), and high (60-87 FID 50 ). To determine the dose at which the ferrets inoculated with PN99 virus become contagious, two naïve ferrets for each transmission experiment were placed either in the same cage as inoculated ferrets or in an adjacent cage with a perforated adjoining wall, to assess contact or respiratory droplet transmission, respectively, as described (8).…”

“…Serial dilutions of virus were administered to two to six ferrets each IN or by AR to assess infectivity, and disease progression was evaluated at all doses. Inoculations were grouped into dose categories: for IN doses, low (1.2 FID 50 ); intermediate (12 FID 50 ); and high (62-115 FID 50 ); for AR-presented doses, low (1.5-1.8 FID 50 ); intermediate (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) ), and high (60-87 FID 50 ). Additionally, a 100 FID 50 IN dose or 66-87 FID 50 AR-presented dose was administered to three ferrets each, and at 3 dpi virus titers in tissues were determined.…”

“…Serial dilutions of virus were administered either IN or by AR to two to five ferrets each until an end point was reached, and no virus shedding was detected in nasal washes. Once the 50% ferret infectious dose (FID 50 ) values were determined retrospectively for each inoculation, doses were grouped into dose categories as follows: for IN doses, low (1.8-2.3 FID 50 ), intermediate (23 FID 50 ), and high (100-227 FID 50 ); and for ARpresented doses, low (1.5-2.2 FID 50 ), intermediate (15)(16)(17)(18)(19)(20)(21)(22) ), and high (395-526 FID 50 ). At each dose, transmission was assessed by placing two naïve ferrets in contact with inoculated animals, either in the same cage for contact transmission or in an adjacent cage with a perforated side wall for respiratory droplet transmission.…”

Complexities in Ferret Influenza Virus Pathogenesis and Transmission Models

“…It is understandable that the way a person is exposed to influenza virus could influence the resulting disease that occurs once infection is initiated. For example, recent studies describing inoculation of ferrets by the aerosol exposure route (either whole-body or nose-only exposure) have been driven by efforts to more closely mimic a typical human airborne exposure to virus in this species (23,24), and studies examining the potential of virus infection of ferrets following consumption of infected meat have met a public health need (25,26). Modulation of the inoculation route can affect where the virus is deposited following exposure (24,27), where initial virus replication takes place (22,26), and the timing and extent of virus dissemination to other tissues (26,28,29).…”