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Molecular Basis for High
Virulence of Hong Kong H5N1
Inßuenza A Viruses
Masato Hatta,1 Peng Gao,1 Peter Halfmann,1
Yoshihiro Kawaoka1,2*
In 1997, an H5N1 inßuenza A virus was transmitted from birds to humans in
Hong Kong, killing 6 of the 18 people infected. When mice were infected with
the human isolates, two virulence groups became apparent. Using reverse
genetics, we showed that a mutation at position 627 in the PB2 protein
inßuenced the outcome of infection in mice. Moreover, high cleavability of the
hemagglutinin glycoprotein was an essential requirement for lethal infection.
An outbreak of H5N1 influenza A virus in
Hong Kong in 1997 resulted in 6 deaths among
18 people infected (1–3). The virulence seen in
mice when infected with these human isolates
showed some correspondence with the severity
of disease in adult patients, although there are
exceptions (3–7). To determine the molecular
basis for the difference in virulence among
Hong Kong H5N1 viruses, we generated reassortants
between virulent and avirulent viruses
(Fig. 1) using a plasmid-based reverse genetics
system (8). First, we constructed plasmids in
which a human RNA polymerase I promoter
and a mouse polymerase I terminator flanked
cDNAs for the full-length RNAs of A/Hong
Kong/483/97 (HK483) (which caused lethal
systemic infection in mice) or A/Hong Kong/
486/97 (HK486) (which produced nonlethal respiratory
infection) viruses (9). Two forms of
the HA gene were identified in our HK486
virus stock: one possessing Ser at position 227
(H3 numbering) (486HA227S) and another
with Ile at position 227 (486HA227I). Using
these plasmids, we produced HK483 and
HK486 transfectant viruses (10)—designated
HK483RG, HK486RG(HA-227S), and HK-
486RG(HA-227I)—and inoculated them into
Madin-Derby canine kidney (MDCK) cells to
produce virus stocks.
Figure 1 and Table 1 compare the pathogenicity
of the transfectants, based on the
virus dose lethal to 50% of infected mice
(50%...