Modification of specific regions of the human cytomegalovirus genome during in vitro passage

Different regions of the human cytomegalovirus strain AD-169 DNA molecule were tested for stability using cloned Hind III fragments. Results indicate that insertion, deletion and/or rearrangement of nucleotide sequences occur at a relatively high frequency in all parts of the human cytomegalovirus genome during in vitro passage.

previously described (18).Viral DNA was extracted from infected cells using the technique developed for herpes simplex virus (19).Random Hind III fragments obtained after complete digestion of the crude DNA preparations were cloned in the plasmid vector pAT153 (3.7 kb) according to standard procedures.as previously described in detail (20).More than 2000 recombinant plasmids were rapidly screened for the presence of DNA inserts between 6 and 14 kb using agarose gel electrophoresis (21).Plasmids carrying such pieces of viral DNA were cleaved with restriction enzymes EcoRl and PsU in the appropriate buffer and temperature conditions, then run in 1% agarose for 16 h at 50 V.

RESULTS
The human cytomegalovirus Hind III-L (12.0 kb).-M (11.0 kb).-N (9.0 kb).-0 (8.9 kb) and -P (8.0 kb) restriction fragments represent 21% of the viral genome (230 kb), and they are found between positions 0.23 and 0.76 on the DNA cleavage map as shown in Figure 1.Sixteen recombinant plasmids carrying the Hind III-P fragment of human cytomegalovirus were obtained.Two of these (pLCC873 and pLCC 1430) contained additional -M (11 .0 kb) , -N (9.0 kb) , -0 (8.9 kb) and -P (8.0 kb) restrictionjrag ments within the human cytomegalovirus genome.Thickened areas represent the inverted repeats of the long and short invertible genome components (22).The point at which inversion occurs is indicated by the dotted line bases.Results shown in Figure 2 (lanes 3 and 7) indicate that in these cases, the cloned human cytomegalovirus Hind III -P fragment was about 100 and 500 base pairs longer than usual (compared to other lanes).Insertion or deletion of genetic material (up to 6 kb) was also detected in an equivalent number of recombinant plasmids canying the human cytomegalovirus Hind III-L, -M and -N fragments (results not shown).No differences were found, however, in the number and/or size of restriction fragments obtained after digestion of nine recombinant plasmids with the Hind III-0 fragment as an insert.

DISCUSSION
The DNA of a particular strain of human cytomegalovirus was considered until now as structurally stable during in vitro passage.It is well known, however, that human cytomegalovirus genomic variants may appear after less than five tissue culture passages (4) .Human cytomegalovirus subclones may be characterized by very similar restriction pattems, but 'fuzzy modifications' in minor bands are sometimes reported (1,2,4,10).As suggested by the present results, insertion, deletion and/or rearrangement of nucleotide sequences occur in all parts of the human cytomegalovirus genome and at relatively high frequencies, but unless the newly generated viral DNA molecule replicates in the cell faster than the original one, only minor changes will be observed in the restriction pattems of the virus initially used for infection.
Unnoticed before among a whole population of normal digestion products, the modified human cytomegalovirus DNA sequence finally appeared through the cloning amplification process.Most of the observed changes in viral DNA will probably lead to gene inactivation and consequently to a defective virus.Occasionally, however, the possible introduction of a strong promoter in front of an important functional gene or the loss of a key control DNA sequence may result in a virus with improved growth properties under specific culture conditions.In this case, the original virus would Figure 2) Restriction profiles of plasmids pLCA223 {1), pLCA365 (2), pLCC783 (3), pLCC803 (4), pLCC1211 ( 5), pLCC1422 (6) and pLCC1430 (7).The 1-kbp ladder from Bethesda Research Laboratories was used as a marker (M) .Electrophoresis was carried out in l% agarose for 16 h at 50 V.pLCA223 and pLC365 represent p lasmids carrying the normal human cytomegalovirus Hind III-P fragment in the two possible orientations be in a minority situation at the end of the incubation period, which means new DNA restriction pattems to observe.
The stock of human cytomegalovirus strain AD-169 used in the present experiments was first plaque-purified and then maintained by passaging at low multiplicity (multiplicity of infection not more than 0 .1) to avoid variants and to mimic clinical isolates in their very nature .Only Hind III fragments located at a certain distance from the turbulent, heterologous region separating the long and short segments of the human cytomegalovirus genome (22).were tested for stability.Wildtype and recombinant pAT153 plasmids are known, on the other hand, to be stable in the HB 10 1 (rec A) strain of Escherichia colL Similar experiments done with canine and murine adeno-viruses gave negative results.Based on these observations the possibility that the techniques used may have introduced artefacts not present in the replicated viral DNA seems unlikely.
Many workers have used restriction endonuclease analysis to track human cytomegalovirus transmission, and the results indicate sufficient stability in the virus for valuable conclusions to be drawn ( 1-1 7).Only two of 16 ( 12%) Hind III-P fragments exhibited altered restriction patterns.Thus analysis of the original human cytomegalovirus 'population' would likely give a consistent pattern, as 88% of Hind III-P fragments would have identical restriction endonuclease sites.The authors' recommendation following the present experiments is that, as soon as clinical isolates have to be passaged more than one or two times in cell culture in order to get advanced cytopathic effects, the DNA patterns of the virus should be interpreted with some circumspection.