H SERVICE,
» Sp,

ot HEALTy,
BS &,

 

i

Se DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service
( National Institutes of Health
Cc Bethesda, Maryland 20892
} a

Building
Room 100

(301) 496- 4595

FAX: (301) 496-8312
March 9, 1990

Dr. Joshua Lederberg
President

Rockefeller University
1230 York Avenue

New York, NY 10021

oe one
ne

x.

oN

Ty
a

ny 2 2
22 tt
; fe

 

Dear Josh:

Packaging and expression of a foreign gene by influenza A
virus was recently achieved by Peter Palese and his colleagues
(Cell 57: 1107-1113, 1989). However, the complexity of the
procedures employed and the requirement for infection by a helper
influenza A virus suggests that this approach does not pose a
significant offensive threat at this time. The successful strategy
used by Palese's group included: (1) construction of a chimeric
cDNA containing the sequence for the coding region of a foreign
gene (CAT in this’ instance) fianked by the 3' and _é £@=°S5!
transcriptional, replicative and packaging signals of an influenza
A virus gene, (2) transfection of a cRNA transcript of the cDNA in
the presence of purified influenza A virus polymerase complex
proteins and (3) infection by an influenza A helper virus. It
appeared that the chimeric cRNA was amplified and packaged in
influenza A virus particles which were not infectious but which
could replicate with low efficiency in cells co-infected with a
fully competent influenza A helper virus. The low efficiency of
replication of the recombinant influenza A virus particles and the
requirement for infection with an influenza A helper virus suggests
that in vivo infection by recombinant virus particles and
transmission to contacts are unlikely to occur.

More ominous are the implications of the most recent study by
Palese's group. Previously, it had not been possible to introduce
either genetic information derived from cDNA or site-specific
mutations into the genome of any negative strand RNA virus. Ina
manuscript that I have sponsored for publication in PNAS Peter and
his associates describe the introduction of an engineered influenza
gene, synthesized in vitro from cDNA, into a fully infectious
influenza A virus. This means it should be possible to increase
the virulence of influenza A virus by introduction of specific
mutations at sites known to affect the virulence of avian influenza
A viruses. For example, insertion of 3 to 5 basic amino acids
(arginine or lysine) immediately upstream of the cleavage site of
the hemagglutinin glycoprotein would probably cause a significant
increase in virulence of human influenza A viruses. Cleavage of
the hemagglutinin by host cell protease(s) is required for
infectivity of influenza A virus and any mutation that increases
the efficiency of this cleavage extends cell tropism and increases
virulence. Fortunately, none of the H1, H2 or H3 hemagglutinins
present on influenza A viruses recovered from humans over the past
57 years (since the recovery of the first human influenza A virus
in 1933) possess more than one basic amino acid at the cleavage
site. Also, it may be possible to express chimeric genes that
encode a foreign protein and an influenza A viral protein. There
are several strategies that could be employed to pursue this
objective. Each of these prospects is rather frightening.

Sincerely,

fr

Robert M. Chanock, M.D.

Chief

Laboratory of Infectious Diseases

National Institute of Allergy
and Infectious Diseases

National Institutes of Health

cc: Dr. Thomas Monath
Dr. Alexis Shelokov
Dr. John Steinbruner
Ms. Lynn Rusten