SUMMARYWe provide evidence that (i) variants lacking individual herpes simplex virus type 1 (HSV-1) XbaI sites can be selected following extensive XbaI treatment of the viral DNA and can be recombined to produce HSV-1 variants lacking two of the four sites normally found, (ii) all XbaI sites can be removed from a viable intertypic recombinant HSV genome, (iii) following XbaI treatment, different mutants with deletions (0.15 to 8.8 kb) in the long repeat (TR L or IRL) and long unique regions can be readily isolated, as well as mutants with novel XbaI sites, (iv) several mutants with deletions in one of the repeats (TRL or IRL) have a measurable growth disadvantage in tissue culture.

SUMMARYin the process of generating restriction endonuclease site deletion mutants, we have isolated and characterized three mutants of herpes simplex virus type 2 (HSV-2), strain HG52, with large genomic deletions in Us and TRs. The deleted sequences (7.5 kb) extend from 0-94 map coordinates (m.c.) to 0-99 m.c. and are presumed to contain the HSV-2 gene equivalents of US10, 11 and 12, one copy of immediate early (IE) gene 3 and one copy of an origin of replication (ORls). One of the mutants (HG52X163X12) has a simple deletion whereas in the two others (HG52X 163X14 and HG52X163X21) the deleted sequences have been replaced by inverted duplications of Us/IRs sequences between 0.83 and 0-91 m.c. such that the molecules have short region inverted repeats extended by 6 kb on either side. All three are viable, stable and grow in tissue culture indicating that the polypeptides coded by the HSV-2 genes equivalent to US10, 11 and 12 are n0n-essential for lytic growth in BHK21/C13 cells. In addition the lack of one copy of the HSV-2 equivalent of IE gene 3 and ORIs in HG52X163X12 shows that only one copy of each suffices for viability. However the temperature restriction of the mutants at 38-5 °C suggests that one or more of the polypeptides coded by the deleted sequences may be required in conjunction with another polypeptide(s) for viral growth or stability at the higher temperature. INTRODUCTIONDeletions in one copy of the long repeat sequences of herpes simplex virus type 2 (HSV-2) strain HG52 occur at a frequency of 24~o by passage eight from the original field isolation (Harland & Brown, 1985). The described deletions range in size with the maximum being 9 kb and encompass the entire long repeat with the exception of the 'a' sequences. Other strains of HSV-2 exhibit simdar deletions showing that the HG52 strain is not exceptional. Deletions in the long repeat had been observed previously in intertypic reeombinants when Davison et al. (1981) suggested that they probably arose as a consequence of heterotypic repetitive regions. Our observations show that this could not be the explanation but that deletions within one copy of the long repeats of the HG52 genome are relatively common. These deletion variants do not have a selective disadvantage (Harland & Brown, 1985); thus only one copy of immediate early (IE) gene 1 and the other coding information within the long repeats is sufficient for viable growth in tissue culture. The product of IE gene 1, i.e. Vm,~ IE 110, in conjunction with V mw IE175 has an enhancing effect on early polypeptide transcription (Everett, 1984) but it has not been shown whether V~wlE110 is essential for lytic growth.The high frequency of spontaneous deletions within the long repetitive sequences raises the question of whether similar deletions could occur in the short repetitive sequences of the genome. The repeat (TRs) and unique short (Us) regions of the HSV-l genome have been fully sequenced (Murchie & McGeoch, 1982;McGeoch et al., 1985), The IE3 gene coding for

Histologic diagnosis of lymphoma is far more difficult in the disaggregated cells obtained by percutaneous aspiration of lymph nodes than in tissue sections prepared from excisional biopsy specimens. However, the simplicity, economy, and safety of aspiration biopsy makes this an attractive diagnostic option in certain situations. In the present study, we demonstrate that lymph node aspirates provide material that is both suitable and sufficient for accurately detecting clonal proliferations of B cells by analysis of immunoglobulin gene rearrangements. The rearrangements detected in aspirated tissue serve as clonal markers that can be directly compared with the rearrangements found in histologically confirmed lymphoma removed by open biopsy. The application of gene rearrangements to aspirated material therefore offers a useful method of diagnosing lymphoma, particularly for the purposes of more thorough staging at initial presentation or the evaluation of tissues for possible relapse.

A series of mutants of equine herpesvirus-1 (EHV-1) which contain deletions in non-essential genes was previously characterized in a murine intranasal infection model. One mutant, ED71 which was shown to be attenuated in the model, was further characterized by inoculation into pregnant mice. Despite the attenuation previously reported, intranasal inoculation of pregnant mice resulted in premature parturition and the birth of dead or dying foetuses. Furthermore, mice inoculated before pregnancy with the same mutant, and subsequently challenged 14 days after conception with wild-type virus, were not protected from abortion.

The genome structure of a spontaneously generated HSV-1 strain 17 variant, 1720, has been determined by restriction endonuclease and Southern blot analysis. The short segment of 1720 is unaltered compared to the parental strain 17 genome, whereas the long segment is extensively rearranged. Almost all of TRL (approximately 9.2 kb) has been deleted and consequently IRL is converted into unique sequence. Sequences from approximately 9200 nucleotide position (np) to 97,000 np are present in inverted orientation, covalently bound to sequences in the prototype orientation from approximately 94,000 np to the L/S junction at 126,372 np. Thus, sequences from 94,000 np to 97,000 np are now diploid, with one copy in the normal orientation and location, and the other at the long terminus as an inverted repeat; no inversion of the intervening unique sequences occurs about this novel inverted repeat. In contrast, normal inversions of the long and short segments occur to give four equimolar genomic isomers, indicating that the novel long terminus has gained an "a" sequence. The duplication of sequences between 94,000 np and 97,000 np results in a genome containing two copies of UL43 and one complete and one partial copy each of genes UL42 and UL44 encoding the 65 kD DNA-binding protein and glycoprotein C, respectively. The variant has been shown to grow normally in vitro following high multiplicity infection.