Hot spot mutations in morphological transforming region II (ORF 79) of cytomegalovirus strains causing disease from bone marrow transplant recipients

Nested polymerase chain reaction (PCR) amplifying the morphological transfo rming region II (mtrII) of cytomegalovirus (CMV) has been shown to be usefu l in the detection of CMV DNA in bone marrow transplant (BMT) recipients. H owever, there has never been any report on mutation hot spots and subtypes of this open reading frame. Using primers derived from sequences upstream a nd downstream of mtrII (ORF 79), CMV DNA from peripheral blood leukocytes ( PBL) and conventional CMV culture of 16 BMT recipients were amplified by PC R, cloned into pUC118, and sequenced. The amino acid sequences were predict ed using the standard triplet code. The DNA sequences obtained from direct amplification of CMV in PBL obtained from the 16 patients were 100% identic al to the corresponding ones obtained by amplification of CMV DNA extracted from conventional CMV culture. Within mtrII (ORF 79), hot spot single base mutations were observed at positions +40 (G-->A), +123 (A-->G), +213 (T--> C), and +219 (T-->C). However, because of third base degeneracy, only amino acid 14 was changed from valine to isoleucine in the predicted protein of 13 patients. This corresponded to the hot spot mutation at position +40 (GT C-->ATC), while the rest were silent mutations. An insertion of 3 bases (AC G) was observed in the CMV DNA of 10 patients at positions +91 to +93, lead ing to a threonine insertion at amino acid 31 in these patients. For patien t no. 147 there was a 65 bp deletion in the CMV DNA amplified later in the course of BMT as compared with that early in the course. This gave rise to a frame shift mutation and a change of more than 70% in the predicted amino acid sequence of the protein.