Transmission of a 2009 H1N1 pandemic influenza virus occurs before fever is detected, in the ferret model

Abstract

During the early phase of the 2009 influenza pandemic, attempts were made to contain the spread of the virus. Success of reactive control measures may be compromised if the proportion of transmission that occurs before overt clinical symptoms develop is high. In this study we investigated the timing of transmission of an early prototypic strain of pandemic H1N1 2009 influenza virus in the ferret model. Ferrets are the only animal model in which this can be assessed because they display typical influenza-like clinical signs including fever and sneezing after infection. We assessed transmission from infected animals to sentinels that were placed either in direct contact or in adjacent cages, the latter reflecting the respiratory droplet (RD) transmission route. We found that pre-symptomatic influenza transmission occurred via both contact and respiratory droplet exposure before the earliest clinical sign, fever, developed. Three of 3 animals exposed in direct contact between day 1 and 2 after infection of the donor animals became infected, and 2/3 of the animals exposed at this time period by the RD route acquired the infection, with the third animal becoming seropositive indicating either a low level infection or significant exposure. Moreover, this efficient transmission did not temporally correlate with respiratory symptoms, such as coughs and sneezes, but rather with the peak viral titre in the nose. Indeed respiratory droplet transmission did not occur late in infection, even though this was when sneezing and coughing were most apparent. None of the 3 animals exposed at this time by the RD route became infected and these animals remained seronegative at the end of the experiment. These data have important implications for pandemic planning strategies and suggest that successful containment is highly unlikely for a human-adapted influenza virus that transmits efficiently within a population.

Figure 1. Transmission of pandemic H1N1 between inoculated and co-housed ferrets.

Two ferrets were inoculated with 10⁴ PFU of E195 and 1 day pi a naive sentinel was co-housed with each inoculated donor. Daily nasal washes collected from inoculated (A) and exposed (B) animals were titrated by plaque assay. Ferrets in the same cage are indicated (triangles or squares).

Figure 2. Transmission of influenza virus between co-housed ferrets, at both early and late periods during infection.

Three ferrets were inoculated with 10⁴ PFU of E195. For 30 hours between days 1 and 2 pi each inoculated donor was co-housed with a naive sentinel (exposed group 2). A different group of naive animals were co-housed with the inoculated donors for 30 hours between days 5 and 6 pi (exposed group 3). (A) The core body temperatures of the inoculated donors were continuously monitored both before (above panel) and after (bottom panel) inoculation. The thin horizontal black line indicates baseline temperature (38.5°C) and the horizontal red line indicates fever (39.4°C). (B) The number of sneezes was recorded during a 15 minute observation period for the inoculated animals. Viral titres shed in nasal wash were determined by plaque assay: innoculated (C), exposed group 2 (D) and exposed group 3 (E). Ferrets in the same cage are indicated (red, blue and black). Exposure periods are indicated by the open bars.

Figure 3. Transmission of influenza virus between co-housed ferrets exposed prior to clinical signs.

One donor ferret was inoculated with 10⁴ PFU of E195. For 4 hours between 16 and 20 hours pi the inoculated donor was co-housed with 3 naive sentinels (exposed group 4), and for 4 hours between 24 and 28 hours pi with 2 other naive animals (exposed group 5). (A) The core body temperature of the inoculated donor was monitored by telemetry. The thin horizontal black line indicates baseline temperature (38.5°C) and the horizontal red line indicates fever (39.4°C). (B) The number of sneezes during a 15 minute observation period was recorded for the inoculated ferret. Viral titres shed from the nose were determined by plaque assay for the inoculated (C), exposed group 4 (D) and exposed group 5 (E). Exposure periods are indicated by the open bars.

Figure 4. Respiratory droplet transmission of influenza virus.

Four ferrets were inoculated with 10⁴ PFU of E195-I219K and 1 day pi naive sentinels (Exposed Group 6) were housed in cages adjacent to each donor. (A) The core body temperature of two of the inoculated donors was monitored by telemetry. The thin horizontal black line indicates baseline temperature (38.5°C) and the horizontal red line indicates fever (39.4°C). Virus shed in nasal wash from inoculated (B) and exposed (C) animals was titrated by plaque assay. Ferrets in adjacent cages are indicated (red, blue, black and green).

Figure 5. Respiratory droplet transmission of influenza virus, occurred before but not during clinical signs.

Four ferrets were inoculated with 10⁴ PFU of E195-I219K. For 30 hours between days 1 and 2 pi a naive sentinel (exposed group 7) was housed in an adjacent cage to each inoculated donor. A different group of sentinels (exposed group 8) were exposed to air from the inoculated animals for 30 hours between days 5 and 6 pi. The number of sneezes (A) and coughs (B) in a one hour observation of the inoculated donors were recorded. Viral titres in daily nasal wash were determined by plaque assay: inoculated (C), exposed group 6 (D) and exposed group 7 (E). Ferrets in adjacent cages are indicated (red, blue and black). Exposure periods are indicated by the open bars.