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Federal Register / Vol. 46, No. 126 / Wednesday, July 1, 1981 / Notices 
murine adenovirus strain FL genomes 
can be used as vectors in P2 conditions 
when production of viral particles 
cannot occur (e.g., transformation of 
nonpermissive cells or propagation of an 
unconditionally defective recombinant 
genome in the absence of helper), 
provided the inserted DNA sequences 
are not derived from eukaryotic viruses. 
In the latter case, such experiments will 
be evaluated by NIH on a case-by-case 
basis [45) and will be conducted under 
the prescribed physical and biological 
containment conditions. (See Section 
IV-E-l-b-(3Hc).) 
Ill-C-l-e. All Viral Vectors. 
Ill-C-l-e-(l). Other experiments 
involving eukaryotic virus vectors can 
be done as follows: 
III-C-l-e-(l)-(a). Recombinant DNA 
molecules containing no more than two- 
thirds of the genome of any eukaryotic 
virus [alt viruses from a single Family 
[36) being considered indentical [50)] 
may be propagated and maintained in 
cells in tissue culture using Pi 
containment. For such experiments, it 
must be shown that the cells lack helper 
virus for the specific Families of 
defective viruses being used. The DNA 
may contain fragments of the genomes 
of viruses from more than one Family 
but each fragment must be less than 
two-thirds of a genome. 
III-C-l-e-(l)-(b). Recombinants with 
less than two-thirds of the genome of 
any eukaryotic virus may be rescued 
with helper virus using P2 containment 
if wild type strains of the virus are CDC 
Class 1 or 2 agents, or using P3 
containment if wild type strains of the 
virus are CDC class 3 agents (2). 
Ill-C-l-e-(2). Experiments involving 
the use of other whole or defective virus 
genomes to propagate DNA sequences 
from prekaryotic or eukaryotic 
organisms (and viruses), or as vectors to 
transform nonpermissive cells, will be 
evaluated by NIH on a case-by-case 
basis [45) and will be conducted under 
the prescribed physical and biological 
containment conditions. (See Section 
IV-E-l-b-(3)-(c).) 
NIH will also reveiw on a case-by- 
case basis [45] all experiments involving 
the use of virus vectors in animals and 
will prescribe the physical and 
biological containment conditions 
appropriate for such studies. (See 
Section IV-E-l-b-(3)-(c).) 
Ill-C-l-f. Nonviral Vectors. 
Organelle, plasmid, and chromosomal 
DNAs may be used as vectors. DNA 
recombinants formed between such 
vectors and host DNA, when propagated 
only in that host (or a closely related 
strain of the same species), are exempt 
from these Guidelines (see Section I-E). 
DNA recombinants formed between 
such vectors and noiiviral DNA from 
cells other than the host species require 
only PI physical containment for cells in 
culture since vertebrate cells in tissue 
culture inherently exhibit a very high 
level of containment. Recombinants 
involving viral DNA or experiments 
which require the use of the whole 
animals will be evaluated by NIH on a 
case-by-case basis. (45) 
III-C-2. Invertebrate Host-Vector 
Systems. 
III-C-2-a. Insect Viral Vectors. As 
soon as information becomes available 
on the host range restrictions and on the 
infectivity, persistence, and integration 
of the viral DNA in vertebrate and 
invertebrate cells, experiments involving 
the use of insect viruses to propagate 
DNA sequences will be evaluated by 
NIH on a case-by-case [45) and will be 
conducted under the recommended 
physical containment conditions. (See 
Section IV-E-l-b-(3)-(c).) 
III-C-2-b. Nonviral Vectors. 
Organelle, plasmid, and chromosomal 
DNAs may be.used as vectors. DNA 
recombinants formed between such 
vectors and host DNA, when propagated 
only in that host (or a closely related 
strain of the same species), are exempt 
from these Guidelines (see Section I-E). 
DNA recombinants formed between 
such vectors and DNA from cells other 
than the host species require Pi physical 
containment for invertebrate cells in 
culture since invertebrate cells in culture 
inherently exhibit a very high level of 
containment. Experiments which require 
the use of whole animals will be 
evaluated by NIH on a case-by-case 
basis. [45) 
III-C-3. Plant Viral Host-Vector 
Systems. [48) The DNA plant viruses 
which could currently serve as vectors 
for cloning genes in plants and plant cell 
protoplasts are Cauliflower Mosaic 
Virus (CaMV) and its close relatives 
[2A) which have relaxed circular 
double-stranded DNA genomes with a 
molecular weight of 4.5 X 10 ®, and Bean 
Golden Mosaic Virus (BGMV) and 
related viruses with small (<10® 
daltons) single-stranded DNA genomes. 
CaMV is spread in nature by aphids, in 
which it survives for a few hours. 
Spontaneous mutants of CaMV which 
lack a factor essential for aphid 
transmission arise frequently. BGMV is 
spread in nature by whiteflies, and 
certain other single-stranded DNA plant 
viruses are transmitted by leafhoppers. 
The DNA plant viruses have narrow 
host ranges and are relatively difficult to 
transmit mechanically to plants. For this 
reason, they are most unlikely to be 
accideritally transmitted from spillage of 
purified virus preparations. 
When these viruses are used as 
vectors in intact plants, or propagative 
plant parts, the plants shall be grown 
under Pi conditions — that is, in either a 
limited access greenhouse or plant 
growth cabinet which is insect- 
restrictive, preferably with positive air 
pressure, [2A) and in which an insect 
fumigation regime is maintained. Soil, 
plant pots, and unwanted infected 
materials shall be removed from the 
greenhouse or cabinet in sealed insect- 
proof containers and sterilized. It is not 
necessary to sterilize run-off water from 
the infected plants, as this is not a 
plausible route for secondary infection. 
When the viruses are used as vectors 
tissue cultures or in small plants in 
axenic cultures, no special containment 
is neccessary. Infected plant materials 
which have to be removed from the 
greenhouse or cabinet for futher 
research shall be maintained under 
insect-restrictive conditions. These 
measures provide an entirely adequate 
degree of containment. 
They are similar to those required in 
many countries for licensed handling of 
“exotic” plant viruses. 
The viruses or their DNA may also be 
useful as vectors to introduce genes into 
plant protoplasts. The fragility of plant 
protoplasts combined with the 
properties of the viruses provides 
adequate safety. Since no risk to the 
environment from the use of the DNA 
plant virus/protoplast system is 
envisaged, no special containment is 
necessary, except as described in the 
following paragraph. 
Experiments involving the use of plant 
genomes to propagate DNA sequences 
from eukaryotic viruses will be 
evaluated by NIH on a case-by-case 
basis [45) and will be conducted under 
the prescribed physical and biological 
containment conditions. (See Section 
IV-E-l-b-(3)-(o).) 
IIl-C-4. Plant Host-Vector Systems 
Other than Viruses. (48) Organelle, 
plasmid, and chromosomal DNAs may 
be used as vectors. DNA recombinants 
formed between such vectors and host 
DNA, when propagated only in that host 
(or a closely related strain of the same 
species), are exempt from these 
Guidelines (See Section I-E). DNA 
recombinants formed between such 
vectors and DNA from cells other than 
the host species require P2 physical 
containment. The development of host- 
vector systems that exhibit a high level 
of biological containment, such as those 
using protoplasts or undifferentiated 
cells in culture, permit [2A) a decrease 
in the physical containment to Pi. 
Intact plants or propagative plant 
parts which cannot be grown in a 
[162] 
