Federal Register / Vol. 46, No. 233 / Friday, December 4, 1981 / Notices 
59411 
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. Inverebrate Viral Vectors. 
Experiments involving invertebrate 
virus vectors can be done as follows: 
III-C-2-a-{l). Recombinant DNA 
molecules containing no more than two- 
thirds of the genome of any invertebrate 
virus (all viruses from a single Family 
(36) being considered identical (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 one Family but each fragment 
must be less than two-thirds of a 
genome. 
III-C-2-a-{2). Recombinants with less 
than two-thirds of the genome of any 
invertebrate virus may be rescued with 
helper virus using P2 containment unless 
it is classified by the CDC as a class 3 
agent (1) in which case P3 containment 
is required. 
III-C-2-a-(3). Experiments involving 
the use of other whole or defective virus 
genomes to propagate DNA sequences 
from prokaryotic 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 imder 
the prescribed physical and biological 
containment condtions. (See Section IV- 
E_l_b-(3)-(c).) 
NIH will also review 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).) 
IIl-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 the 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 inherently exhibits 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 coloning 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 difficulty 
to transmit mechanically to pleints. For 
this reason, they are most rmlikely to be 
accidentally 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 imwanted infected 
materials shall be removed from the 
greenhouse or cabinet in sealed iQsect- 
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 in 
tissue cultures or in small plants in 
axenic cultures, no special containment 
is necessary. Infected plant materials 
which have to be removed from the 
greenhouse or cabinet for further 
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)-(c).) 
III-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 
standard P2 laboratory because of their 
large size may be grown under t!ie Pi 
conditions described above in Section 
lII-C-3, except that (i) sterilization of 
run-off water is required where this is a 
plausible route for secondary infection 
and (ii) the standard P2 practices are 
adopted for microbiological work, and 
(iii) negative air pressme should be 
employed in the greenhouse or growth 
chamber when infectious agents are 
used which generate airborne 
propagules. 
III-C-5. Fungal or Similar Lower 
Eukaryotic Host- Vector Systems. 
Certain certified HVl and HV2 host- 
vector systems appear in Appendix D. 
The containment levels for these 
systems are given in the subsections of 
Section UI-A. Other systems in the 
future may be certified as HVl and HV2. 
At the time of certification, they may be 
added to Appendix D (and thus the 
containment levels for their use will be 
those of the subsections of Section III- 
A). Alternatively, at the time of their 
certification, another classification of 
containment levels for experiments 
using them may be assigned by NIH. 
In addition to the experiments 
described above, the following 
experiments may be carried out without 
the eukaryotic host (Host C) having 
been approved as an HVl host: DNA 
from Host C may be inserted into a 
vector and propagated in E. coli K-12. 
Subsequently, this recombinant DNA 
may be returned to Host C and 
propagated there under Pi 
conditions.[43] 
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