Detection of a transforming fragment of herpes simplex virus type 2 in clinical specimens by PCR. The Canadian Women's HIV Study Group

Abstract

A PCR assay for the sensitive detection of a transforming fragment of herpes simplex virus type 2 (HSV-2) was developed. Oligonucleotide primers were selected in Xho-2, a transforming region of the BglII N fragment of HSV-2. The assay reached a sensitivity endpoint of 10 copies of the Xho-2 subfragment and did not show cross-reactivity with other herpesviruses, including HSV-1. All 42 HSV-2 isolates scored positive in the assay. The Xho-2 PCR assay was evaluated with 216 clinical specimens and the results were compared with those of cell culture. The best protocol for processing specimens contained in viral transport medium included a centrifugation step followed by cell lysis. Of the 107 specimens positive for HSV-2 by culture, 105 were PCR positive (sensitivity, 98.1%). For one of the two falsely negative samples, ␤-globin as well as sequences from the HSV-2 DNA polymerase gene could not be amplified. The other sample scored positive in both of these reactions but was indeterminate in duplicate tests by Xho-2 PCR. Two of 109 HSV-2 culture-negative specimens tested positive in the PCR assay (specificity, 98.2%). The latter two samples tested positive in a PCR test for the HSV-2 DNA polymerase gene. This novel tool was shown to be sensitive and specific for HSV-2 sequences and should allow for the investigation of the role of HSV-2 in genital cancers.

Relationship of stable integration of herpes simplex virus‐2 BglII N subfragmentXho2 to malignant transformation of human papillomavirus‐immortalized cervical keratinocytes

“…Although in the previous studies the presence of Xho2 sequences was not specifically investigated, it is, nevertheless, premature to rule out the hit-and-run hypothesis for the following reasons. The prevalence of Xho2 sequences and their expression in malignant cervical lesions must be firmly established before speculating on new possible mechanisms; towards this goal we have designed a highly specific PCR test (Guibinga et al, 1996) that is being used for detection of Xho2 sequences in cervical brushings or formalin-fixed archival material. Although there is no clue as to the function on the Xho2 protein and its exact role in the transformation process, the possibility exists that transient expression of this protein could induce (directly or indirectly) irreversible lesions in the cellular genome and as a consequence alter some critical functions; in this case, transient expression of the sequences would be required for the initiation of the transformation process but not for its maintenance.…”

“…As a consequence, the expression of Xho2-ORF would not be expected to be influenced by any antisense transcription mechanism (Ward et al, 1996). In addition, the alignment of the nucleotides and amino acid sequences of U L 43 and the predicted Xho2 proteins showed less homology within the 25 first residues portion; this allowed development of a PCR test specific for the Xho2 subfragment that has proved useful not only for detection of the encoded mRNA but also for virus subtyping of clinical specimens (Guibinga et al, 1996). Other primers in the Xho2-ORF do not discriminate between the 2 subtypes.…”

“…The proteinase K was heat-inactivated at 95°C for 10 min. Samples were clarified from debris and amplified with Xho-a and Xho-b primers as previously described (Guibinga et al, 1996). The amplified products were hybridized with 32 P-labeled Xho-p internal probe.…”

“…Two micrograms of extracted RNA were reverse-transcribed using as antisense primers either Xbo-b or the oligo-dT with conditions as described by Perkin- Elmer (Norwalk, CT) with the downstream primer Xho-b. The cDNAs were amplified with the primers Xho-a and Xho-b and the PCR products were hybridized with 32 P-labeled Xho-p probe, as described previously (Guibinga et al, 1996). To control the specificity of the reaction, RNA samples were similarly amplified but without the reverse transcription step.…”

“…This investigation was done using RT-PCR with either an oligo-dT or Xho-b antisense primer selected within the ORF sequence and RNAs purified from CX16-2/ Xho1ϩ2,3C, CX16-5/Xho1ϩ2,2C and CX18-1/Xho1ϩ2,2C and HPVGS/Xho2,1M and the non-transfected CX16-2. The cDNAs were amplified with Xho-a and Xho-b primers, which demonstrated a high specificity for the HSV-2 Xho2 subfragment as previously described (Guibinga et al, 1996). The amplified products contained in 3 µl of Xho2 DNA template reaction and 50 µl of RNA test reaction were resolved on 2% agarose gels, transferred onto nylon membranes and hybridized with the Xho-p internal probe (shown in Fig.…”