33160 
NOTICES 
discover phenomena of general cell 
biological significance; techniques will 
be more readily available to elucidate 
the sequence of viral nucleic acids, to 
shed light on the role of viral gene 
products in pathogenicity, and eventu- 
ally. to understand the molecular biol- 
ogy of animal and plant viruses to the 
extent that some bacteriophages are 
now understood. It seems apparent 
that this new information will lead to 
a deeper understanding of viral dis- 
eases and to new ways of combating 
them. In the immediate future the 
ability to obtain useful amounts of 
pure viral genomes and subgenomlc 
fragments that cannot be obtained by 
other means will provide scientists and 
physicians with invaluable and inex- 
pensive diagnostic protein and nucleic 
acid reagents. In the more distant 
future It should be possible to use 
gene cloning techniques to obtain 
large amounts of viral proteins; one 
practical benefit from such develop- 
ments might well be effective and safe 
vaccines for control of diseases caused 
by hepatitis viruses, herpesviruses and 
Influenza viruses and many other vir- 
uses, both known and. as yet, un- 
known. 
In addition to being able to clone 
viral genes in bacteria we are now able 
to envisage using certain animal vir- 
uses as vectors for the propagation of 
foreign genes In animal cells; a similar 
system for exploiting a plant virus, 
cauliflower mosaic virus, to clone for- 
eign genes in plant cells may shortly 
become available. The chief impor- 
tance of animal and plant virus vectors 
Is that they can be used to carry genes 
into cells In which they may be fully 
expressed as well as propagated. By 
using specifically designed viral vec- 
tors It may eventually prove possible 
to deliver a specific gene to specific 
target cells; such techniques have ob- 
vious medical, economic, and agricul- 
tural applications but their realization 
will depend upon a great deal of basic 
research. 
VIRAL CLASSIFICATION SYSTEMS AND 
RECOMBINANT DNA EXPERIMENTATION 
The group extensively discussed the 
current safety procedures for holding 
and handling in the laboratory certain 
animal viruses, in particular those 
likely to be used In cloning experi- 
ments in the foreseeable future, either 
as vectors or as the sources of the nu- 
cleic acids to be cloned. Inevitably the 
recommended safety measures for 
using animal (and plant) viruses in re- 
search vary from country to country. 
In the context of recombinant DNA 
research, which Involves a novel set of 
circumstances, none of the available 
classifications of viruses according to 
the risks they pose is entirely satisfac- 
tory. A list of animal viruses was 
therefore prepared and the viruses 
were ranked according to their known 
hazard on the basis of; (a) The sever- 
ity of human disease that they can 
cause, particularly in persons exposed 
in the laboratory; (b) their potential 
for infecting laboratory workers; (c) 
the risk that a laboratory infection 
might result in spread to the commu- 
nity; and (d) the impact such spread 
might have on the community or envi- 
ronment (table 1). In this list four bac- 
teria and one rickettsial agent with 
different pathogenic potentials have 
also been included as a frame of refer- 
ence for the 22 viruses Identified. Be- 
cause of time constraints, many 
animal viruses, particularly those of 
agricultural and veterinary impor- 
tance. were not included in the table. 
Although not comprehensive, the list 
contains most of those animal viruses 
that have been previously mentioned 
In the context of recombinant DNA 
experiments. 
This list can also serve the very 
useful function of a reference scale, fa- 
miliar to both microbiologists and cli- 
nicians. for expressing the degree of 
concern that a given conjectural 
hazard may engender, by comparison 
to a known biohazard. 
CLONING VIRAL DNA'S IN E. COLI K-ll 
The cloning of viral DNA’s and 
cDNA's in E. coli K 12 using EK1 and 
EK2 plasmid and lambda phage vec- 
tors was discussed in light of the con- 
clusions of the Falmouth meeting that 
E. coli K-12 is not pathogenic and 
does not efficiently colonize the verte- 
brate digestive tract (Oorbach. 1978). 
Not for want of trying, the partici- 
pants were unable to envisage a se- 
quence of events which could occur 
with significant probability that would 
allow E. coli carrying either whole 
DNA genomes of certain viruses or 
subgenomlc fragments of virtually any 
virus to lead to disease. The question 
was also raised as to whether or not, in 
the extremely remote possibility that 
all of the biological and physical con- 
tainment barriers broke down. Intesti- 
nal bacteria carrying cloned whole 
viral genomes might bypass the natu- 
ral barriers to infection by the virus 
particle. As summarized in the follow- 
ing section, the group concluded that 
the probability that K-12 organisms 
carrying viral DNA Inserts could repre- 
sent a significant hazard to the com- 
munity was so small as to be of no 
practical consequence. 
Risk assessment analysis for cloning 
viral DNA in E. coli K-12.' The follow- 
ing is a summary of the workshop dis- 
cussions dealing with possible mecha- 
nisms of risk resulting from the clon- 
ing of genetic material of eukaryotic 
viruses (i.e., viruses of animals, plants. 
'This portion of the report was prepared 
subsequent to the U.S.-EMBO workshop by 
Drs. Martin and Rowe. 
or lower eukaryotes) in E. coli vector 
systems. 
We started with the extremely un- 
likely "worst case" assumption that a 
recombinant molecule containing eu- 
karyotic viral genome sequences has in 
some way become established in wild 
type E. coli and has thereby become 
disseminated throughout the bowel 
flora of vertebrates. Given this hypo- 
thetical set of conditions, what conse- 
quences could be envisaged? For the 
purposes of analysis the discussions fo- 
cused on two issues. 
First: The mechanisms by which the 
viral nucleic acid might gain access to 
cells of the host; and 
Second: The nature of the inserted 
viral sequences. 
Access of the viral genome to cells of 
the vertebrate host might conceivably 
result from virus particles formed 
within the bacterium or from release 
of viral nucleic acid into the proximity 
of. or into, host cells; this could occur 
either in the intestine or at the site of 
extralntestlnal E. coli Infection. 
Production of infectious virus parti- 
cles by bacteria carrying the recombin- 
ant DNA molecules was considered to 
be virtually impossible, regardless of 
the completeness of the viral genome 
or the nature of the eukaryotic virus 
from which it was derived. The basis 
for this high degree of assurance is in 
large part our great understanding of 
the molecular biology of virus replica- 
tion. The nature of regulation of gene 
expression In prokaryotes is clearly 
different from that in eukaryotes as 
particularly exemplified by the ab- 
sence in prokaryotes of RNA splicing 
mechanisms (Berget el al.. 1977; Aloni 
et al.. 1977; Lavl and Groner. 1977; 
Mellon and Duesberg. 1977; Chow et 
al.. 1977; Klessig, 1977; Dunn and Has- 
sell. 1977), RNA capping (Moore. 1966; 
Stavis and August. 1970; Blattner and 
Dahlberg, 1972; Matzels. 1973; Wei and 
Moss, 1975; Furuichi et al., 1975a; 
Keith and Fraenkel-Conrat. 1975; 
Abraham et al., 1975; Furuichi et al., 
1975b; Dubln and Taylor, 1975; Perry 
and Scherrer, 1975; Moss and Koczot, 
1976) and differences in messenger 
RNA biogenesis. polyadenylatlon 
(Kates and Beeson. 1970; Darnell et 
al., 1971; Mendecki et al.. 1972; Phllip- 
son et al., 1971; Weinberg et al.. 1972; 
Ehrenfeld and Summers. 1972; Prid- 
gen and Kingsbury. 1972), and riboso- 
mal binding sites (Shine and Dalgamo, 
1974; Steitz and Jakes. 1975; Hagen- 
buchle et al.. 1978). The expression of 
animal viral mRNA in E. coli transla- 
tion systems Is not accurate; in one 
well studied system, poliovirus, inter- 
nal initiation signals are read which 
result in premature chain termination 
(Rekosh et al.. 1970). Thus neither the 
synthesis of proper mRNA nor its 
translation into viral protein is likely 
to occur in E. coli. 
FEDERAL REGISTER, VOL 43, NO. 144 — FRIDAY, JULY 28, 1978 
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