NOTICES 
33161 
Virus replication requires the regu- 
lated and coordinated function of mul- 
tiple enzyme systems derived from 
both the host and the viral genomes; it 
seems most unlikely that any prokar- 
yote contains the complement of en- 
zymes required for the synthesis of an 
infectious animal virus. Animal viruses 
have evolved to be adapted to replica- 
tion in eukaryotic cells; except for the 
instances where they have coevolved 
to replicate in insect vectors, animal 
and plant viruses show a high degree 
of specificity for cells of a particular 
class or species of host cell or even a 
particular differentiated cell type. 
Further, there is no verified example 
of the replication of a virus of eukar- 
yotes in bacteria or conversely, the 
replication of a bacterial virus in any 
eukaryote. Consequently, since it was 
not considered possible for recombin- 
ant DNA containing a viral chromo- 
some tc produce intact virus particles 
in E. coli, the group felt secure in fo- 
cusing on models involving host cell 
exposure to viral nucleic acid rather 
than viral particles. 
In a model in which recombinant 
DNA-containing organisms are con- 
fined to the lumen of the intestine, 
the possibility of viral RNA or DNA 
gaining access to mucosal cells is ex- 
tremely remote. First: The large 
amounts of nucleases in the intestinal 
contents would rapidly destroy the re- 
combinant molecules IMaturin and 
Curtiss. Is*?':;. Second; The efficiency 
•of infection of cells by viral nucleic 
acid molecules, even in the presence of 
chemical potentiators, is extremely 
low, both in vitro and in vivo lElllem 
and Colter, 1961; Amstey and Park- 
man, 1956; McCutchan and Pagano, 
1968: Graham and van der Eb, 1973; 
Israel et ah, 1978a,. Third: Since 
animal experiments indicate that po- 
lyoma viral I >NA cannot initiate infec- 
tion when administered by the oral or 
nasal routes (Israel et al., 1978a), it is 
likely that nucleic acids cannot infect 
across mucous membranes. 
Consequently, the only model to 
which serious attention must be di- 
rected is one in which the hypotheti- 
cal bacterium carrying the viral re- 
combinant DNA gains access to the 
body tissues. Two cases can be consid- 
ered. With minor transgressions of the 
intestinal mucosa that allow brief pen- 
etration of organisms into the intesti- 
nal wall, lymphaties, or the protal cir- 
culation, the bacteria would be ingest- 
ed by phagocytes. In phagocytes, bac- 
teria are lysed and their nucleic acid is 
released in the nuclease-rich lyso- 
somes; the effectiveness of the lysoso- 
mal enzymes virtual] guarantees that 
no nucleic acid could survive to cause 
infection (Bensch et al„ 1964; Carrara 
and Bernard! [1968]; Arsenis et al„ 
1970). 
The second case, extraintestinal in- 
fection such as urinary tract or surgi- 
cal wound infection, deserves serious 
consideration, but it must be pointed 
our that this would occur in only a 
fraction of individuals. In this context 
the consequences of the infection 
would be a function of the nature of 
the viral DNA inserted in the recom- 
binant DNA molecule. We considered 
the following classes of DNA inserts: 
(1) Subgenomic segments of nononco- 
genic viruses; (2) transforming seg- 
ments of oncogenic viruses; (3) cDNA 
prepared from the genome of segment- 
ed RNA viruses; (4) cDNA copies con- 
taining the complete viral genome of 
nonsegmented RNA viruses and (5) 
complete DNA viral genomes. The 
workshop participants reached the 
conclusion that viral inserts should be 
thought in terms of three classes of 
risk: Those for which there was no 
risk of a harmful outcome, and those 
for which a possibility of harm, how- 
ever remote, could be envisioned. The 
latter were then divided into those sce- 
narios which may well in reality be im- 
possible, and those which are felt to be 
possible. 
Those inserts for which the group 
could not construct any realistic harm- 
ful scenario were: (1) Subgenomic seg- 
ments of nontransforming RNA or 
DNA viruses; (2) cDNA copies from 
RNA viruses with segmented viral gen- 
omes and (3) cDNA’s of the complete 
genome of negative strand RNA vir- 
uses. 
'll Subgenomic segments 'meaning 
small portions of the viral genome 
lacking genes needed for replication of 
the virus; of nontransforming viruses 
are considered to be harmless because 
of the absence of any known example 
of an individual viral encoded protein 
which can act exogenously on cells. 
With the exception of some glycopro- 
teins when added to cell cultures in 
high concentrations (Scheid and 
Choppin, 1974; McSharry and Chop- 
pin. 1978), viral proteins do not induce 
cell damage from without. 
(2) Reverse tr ans cripts of RNA vir- 
uses with segmented genomes cannot 
be envisaged as carrying any risk of 
producing infectious virus even when 
the cDNA is made from unfractionat- 
ed nucleic acid preparations. It would 
be virtually impossible to ligate to- 
gether a complete DNA copy, and if 
this ever did occur, we cannot envisage 
any way that the proper length RNA 
genome segments could be transcribed 
therefrom. 
(3) Cloning of reverse transcripts of 
negative strand RNA viruses is viewed 
as being free of risk. With these vir- 
uses, the process of viral mRNA and 
genomic RNA synthesis is complex, 
and the Workshop participants could 
not envisage a set of circumstances in 
which RNA segments, transcribed 
from a DNA template, could eventuate 
in the synthesis of progeny virus. This 
is based on the fact that the nucleic 
acid of negative strand RNA viruses 
(either the plus or min us strand) has 
never been shown to be infectious for 
cells (Kingsbury, 1966: Baltimore et 
al., 1970; Wagner, 1975) presumably 
because of their unique molecular biol- 
ogy. Infection by negative strand vir- 
uses requires the activity of the virion 
associated transcriptase (Baltimore et 
al., 1970; Cormack et aL, 1971; Szilagyi 
and Pringle, 1972); this enzyme cata- 
lyzes the synthesis of multiple (seg- 
mented) functional plus strand mRNA 
molecules from the input min us 
strand (Bratt and Robinson, 1967; 
Huang et al., 1970; Mudd and Sum- 
mers, 1970; East and Kingsbury, 1971; 
Weiss and Bratt, 1976; Freeman et al., 
1977). A polypeptide specified by one 
of these mRNAs then modifies the 
virion transcriptase to function as a 
replicase mediating the synthesis of a 
complete unsegmented plus strand of 
RNA, the template for synthesis of 
progeny minus stand RNA molecules. 
The minus strand has no messenger 
function (Huang et aL, 1970; Grubman 
and Summers, 1973; Kingsbury, 1973; 
Morrison et al„ 1974). Thus, to initiate 
the infectious process both a full 
length RNA transcript (to serve as 
template) and the segmented plus 
strand transcripts, capped for function 
as mRNA, would be required. This 
would necessitate either the transcrip- 
tion of both DNA strands by cellular 
RNA polymerase or the synthesis of a 
full length transcript as well as prop- 
erly terminated, segmented transcripts 
from the minus strand DNA. These 
are obviously extremely unlikely 
events. 
There were two classes in which it 
was considered that a risk scenario 
might be constructed but which might 
indeed be impossible; these involve the 
cloning of the transforming segments 
of DNA viruses or of transforming re- 
troviruses, and the cloning of complete 
reverse transcripts of plus-strand RNA 
■viruses. The model involving subgeno- 
mic, transforming segments postulates 
release of recombinant DNA molecules 
by lysis of E. coli in the tissues, and in- 
tegration of the transforming gene 
segment into the DNA of host cells. 
While this eventuality cannot be ruled 
out, it was considered to have a very 
low probability in view of the ineffi- 
ciency of transforming cells with viral 
nucleic acid (Aaronson and Martin, 
1970; Graham et al., 1974; Abrahams 
et al., 1975) and the fact that integra- 
tion of transforming DNA would occur 
in only a few cells in any one individu- 
al and, in the presence of competent 
immuno- surveillance, would be most 
unlikely to result in a tumor (Habel, 
1961; Sjogren, 1964; Sjogren et al„ 
1967; Costa et al., 1977). Transplanta- 
FEDERA.L REGISTER, VOL. 43, NO. 146— FRIDAY, JULY 28, 1978 
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