Diary Prequel Package — page 272

Fouchier took these 3 mutations and inserted them into a wild type (WT) HPH5N1 (see above). This technique is widely used in molecular virology. He then passaged the virus 10 times in ferrets. "Passaged" means inserting the virus into the nose of the animal, getting the animal to get infected, then taking virus from the nasal passages of the infected animal and inserting it into the nasal passage of the next animal in the sequence, and so on. The endpoint that is aimed at is to get a virus that is then able to be transmitted by aerosol (i.e. not relying on direct insertion into the nasal passage). Since ferrets sneeze when they are infected, one determines "aerosol transmission" by housing an infected ferret in a cage next to a separate cage of an uninfected ferret that is constructed such that an aerosol can get from one cage to the other due to "holes" in the barriers between cages. The virus resulting from the passaging was able to be transmitted by aerosol and was a "quasi-species", i.e. it contained different "versions" of the mutated virus. The viruses that were transmitted by aerosol were then sequenced and all of these viruses had 5 mutations in common: the 3 mutations that were inserted by reverse genetics and 2 additional ones. In addition, the viruses had 4 to 7 variable mutations (this latter sequencing information was not ready for reporting in the original manuscript). This virus isolate was used in the pathogenesis studies reported in the manuscript. It was the isolate with the minimal number of mutations (9 mutations - 5 common mutations + 4 variable mutations). After submission of the manuscript the authors took a wild-type H5N1 clone and inserted this minimal number of mutations to develop a virus that was transmissible without the need for passage. The pathogenesis studies with this cloned virus were identical to those of the isolate reported in the manuscript (see below). It is EXTREMELY IMPORTANT to point out that the animals that got infected by aerosolized virus described above did not die nor did they get sick. They were followed for 14 days. A few got minor flu-like symptoms, all recovered, they then were sacrificed since logistically the author could not house them indefinitely. Thus, the "engineered" virus that was made transmissible, did not kill or make the animals sick when exposed in the manner that flu in transmitted in humans. PATHOGENICITY. The classic way that influenza virus pathogenicity is established (welldescribed in the literature) for the ferret model is by direct insertion of the virus into the nasal cavity and/or directly into the trachea. Fouchier did both nasal and tracheal insertion of the viruses. In the nasal insertion studies, 106 (1 million) infectious doses of virus is inserted into the nose. This is a dose that far exceeds a dose that one gets through aerosolized virus (i.e. sneezing). When this dose of the pandemic 2009 H1N1 virus (not considered a particularly pathogenic virus, particularly in people who have been exposed to H1N1 over years by infection or vaccination - children who are naïve to this virus have had more problems) is inserted into the ferret, the animals develop flu-like symptoms but do not die. When HPH5N1 is inserted the animals essentially all get neurovirulence, they recover, none die. When the modified (aerosoltransmitted) H5N1 virus is inserted, there is some neurovirulence, but actually less neurovirulence than with the wild typeH5N1. Again, all recover and none die.