33168 
NOTICES 
REVIEW or THE U.S.-EMBO WORKSHOP 
REPORT 
The working group discussed the 
U.S.-EMBO workshop report section 
by section and recommended the fol- 
lowing amendments: 
(1) Page 10, line 17— delete "then 
modifies" and insert "Is thought to 
modify.’’ 
(2) Page 16. lines 6-8— The working 
group did not understand the scientif- 
ic basis behind this short paragraph 
which singled out viroids from other 
RNA plant viruses and which would 
postpone work in this area. Deletion of 
these three lines was recommended 
following a discussion about the poten- 
tial risks attending the cloning of 
cDNA copies of viroids in E col i K12. 
(3) Table 1. 
(a) Yellow fever virus— Add ”L (if 
vaccinated)" under Risk of Laboratory 
Infection and change "(H)" to 
under Community or Environmental 
Impact. 
(b) Epstein Barr Virus— Change 
"2 + " to ”0-2 ♦ " under Severity of 
Human Disease. 
After reviewing the U.S.-EMBO 
report the working group unanimously 
endorsed: (1) The classification of vir- 
uses with respect to their ability to 
cause disease in laboratory workers 
and their Impact on the environment; 
(2) The analysis and recommendations 
for cloning viral DNA'i in E. coli K12; 
and (3) The analysis and recommenda- 
tions tor the use of viral DNA's as vec- 
tors in eukaryotic cells. 
IMPLEMENTATION or THE U.S -EMBO 
REPORT 
A. Cloning Eukaryotic Viral DNA in 
E. coli K12 Host- Vector Systems. 
After considerable discussion, the 
working group recommended physical 
and biological containment conditions 
for experiments involving the cloning 
of viral DNA in E. coli K12 as shown 
In table L The group classified the 
viral DNA for such experiments in 
three categories: (1) Virion DNA of 
DNA viruses; (2) cDNA copies of virion 
RNA of RNA viruses, and (3) Intracel- 
lular forms of viral DNA Including in- 
tegrated genomes, and DNA from pro- 
ductively infected cells. The viral DNA 
to be cloned was then subclassified 
into whether it represented the entire 
viral genome or a purified subgenomlc 
segment thereof. Subgenomlc seg- 
ments were further subdivided on the 
basis of whether they contained Intact 
transforming genes or not (see table 
1). In addition, the working group also 
assigned physical and biological con- 
tainment levels for cDNA copies of cel- 
lular mRNA's for each class of viral 
DNA insert (see table 1). 
1. In Its deliberations, the working 
group was impressed with the safe- 
guards afforded by a ban on mouth-pi- 
petting for recombinant DNA experi- 
ments Involving E. coli K12 host-vec- 
tors. The group felt that the only 
plausible way E. coli K12 could gain 
entry into laboratory workers was by 
oral Ingestion. The analysis contained 
in the U.S.-EMBO report was predi- 
cated on the remote possibility that E. 
coli K12. containing eukaryotic viral 
DNA. would be swallowed and the 
viral DNA insert would be delivered to 
a tissue in the body which ordinarily 
would be inaccessible to the virus. A 
prohibition of mouth pipetting would 
clearly prevent this sequence of events 
from even beginning. The working 
group therefore recommended that no 
mouth pipetting be allowed at any 
level of physical containment (includ- 
ing PI) when working w ih E. coli 
K12. 
2. The group was struck by the in- 
herent safety afforded by nonmobill- 
zable plasmids and felt they represent- 
ed an additional level of containment 
when used with E. coli K12. Accord- 
ingly. the working group recommend- 
ed the use of E. coli K12 in conjunc- 
tion with non-moblllzable plasmid vec- 
tors (referred to in table 1 as EK1NM) 
for several categories of experiments. 
3. The virus working group exten- 
sively discussed the use of class III 
(CDC) and "moderately oncogenic" 
(NCI) viral DNA’s In E. coli K12 host 
vector systems. The group concluded 
that vesicular stomatitis virus (VSV), 
which is a widely studied negative- 
strand RNA virus, is classified as a 
class III agent because of its ability to 
produce disease in animals: VSV is not 
an important human pathogen (see 
table 1 of U.S -EMBO report). The 
working group agreed that VSV pos- 
sessed all of the safety features of 
other negative-strand RNA viruses 
(see U.S.-EMBO report) and strongly 
recommended that cloning of VSV 
cDNA be permitted because of Its im- 
portance as a model virus in studies of 
the molecular biology of virus Infec- 
tion. The working group also consid- 
ered the 9 viruses classified as moder- 
ately oncogenic by NCI (Appendix B) 
and concluded that the only potential 
biohazard associated with their use in- 
volved the propagation of the viruses 
themselves during the preparation of 
reagents/substrates for use in recom- 
binant DNA experiments. None of 
these agents produces human disease 
or has been associated with human 
malignancy. The virus working group 
endorsed the Inclusion of these agents 
as sources of eukaryotic viral DNA for 
cloning in E. coli K12 and recommend- 
ed that NCI guidelines be followed for 
work involving the viruses themselves. 
B. Viral DNA Vectors in Eukaryotic 
Cells. 
The working group discussed the 
physical containment levels appropri- 
ate for different eukaryotic viral DNA 
vectors and prepared a list of four 
animal viruses and two plant viruses as 
candidate vectors. In each case the 
group made recommendations regard- 
ing physical containment conditions 
for productive or non-productive virus- 
cell interactions. The virus working 
group, like the participants of the 
U.S.-EMBO workshop, were concerned 
about alterations in host range and 
tissue troplsm that might occur fol- 
lowing the insertion of foreign viral 
DNA sequences into eukaryotic viral 
DNA vectors and were unable to speci- 
fy containment levels for this type of 
DNA recombinant. The group agreed 
that experiments of this type could 
yield useful information about viral 
pathogenesis and recommended that 
each be evaluated by the recombinant 
DNA Molecule Program Advisory 
Commitee on a "case-by-case" (CBC) 
basis. The working group also recog- 
nized Its inability to identify all possi- 
ble viral vectors and therefore includ- 
ed a category which encompassed 
other potential viral DNA vectors 
whose use could also be evaluated on a 
"case-by-case" basis. 
The virus working group regarded 
the use of DNA vectors prepared from 
CaMV and BOMV as posing virtually 
no biohazard to plants or the ecosys- 
tem and recommended a minimum of 
physical containment. The group ex- 
tensively discussed the use of baculo- 
vlrus DNA as vectors, and. despite 
their certification as registered pesti- 
cides. considered the available infor- 
mation about their host range, persis- 
tence. and basic biology to be too rudi- 
mentary at the present time. A "case- 
by-case" evaluation was therefore rec- 
ommended. 
The virus working group unanimous- 
ly endorsed the elimination of the re- 
quirement pertaining to the functional 
anatomy of viral DNA vectors (page 
49602. third column. (Hi)) since these 
features are related in no way to the 
inherent safety of a potential vector. 
These criteria for candidate vectors 
were retained in the final recommen- 
dations. however, as desirable, al- 
though not required features. 
It should be noted that the recom- 
mendations of the virus working group 
impact on two other sections of the 
Revised Guidelines for Recombinant 
DNA Research. 
(1) Section III B la(l)(g) can be 
eliminated because it is now covered in 
section III B lb(l). 
(2) Section III A should be amended 
to read "pathogenic organisms in 
classes 3, 4, and 5 except vesicular sto- 
matitis virus;" the prohibition of mod- 
erate risk oncogenic viruses should be 
deleted. 
The recommendations made by the 
virus working group were based on the 
best and most current available scien- 
tific considerations. The containment 
levels proposed for cloning eukaryotic 
FEDERAL REGISTER. VOl. 43. NO. 144 — FRIDAY, JULY 2t. 1971 
[ 129 ] 
