Unusual immunoglobulin and T-cell receptor gene rearrangement patterns in acute lymphoblastic leukemias.

Immunoglobulin (Ig) and T-cell receptor (TCR) genes are rearranged in virtually all acute lymphoblastic leukemia (ALL) cases. However, the recombination patterns display several unusual features as compared to normal lymphoid counterparts. Cross-lineage gene rearrangements occur in more than 90% of precursor-B-ALL and in approximately 20% of T-ALL, whereas they are rare in normal lymphocytes. Approximately 25-30% of the Ig and TCR gene rearrangements at diagnosis are oligoclonal, and can undergo continuing or secondary recombination events during the disease course. Based on our extensive molecular studies we hypothesize that the unusual Ig and TCR gene rearrangements in ALL occur as an early postoncogenic event resulting from the continuing V(D)J recombinase activity on accessible gene loci. This hypothesis is on the one hand supported by the virtual absence of cross-lineage gene rearrangements in normal lymphocytes and mature lymphoid malignancies and on the other hand by the presence of oligoclonality and secondary Ig and TCR gene rearrangements in ALL.

T cell receptor gamma (TCRG) gene rearrangements in T cell acute lymphoblastic leukemia refelct 'end-stage' recombinations: implications for minimal residual disease monitoring.

The T cell receptor gamma (TCRG) gene configuration was established in a large series of 126 T cell acute lymphoblastic leukemia (T-ALL) patients using combined Southern blotting (SB) and heteroduplex PCR analyses. The vast majority of TALL (96%) displayed clonal TCRG gene rearrangements, with biallelic recombination in 91% of patients. A small immature subgroup of CD3- T-ALL (n = 5) had both TCRG genes in germline configuration, three of them having also germline TCRD genes. In five patients (4%) combined SB and PCR results indicated oligoclonality. In five rearrangements detected by SB, the Vgamma gene segment could not be identified suggesting illegitimate recombination. Altogether, 83% of TCRG gene rearrangements involved either the most upstream Vgamma2 gene (including four cases with interstitial deletion of 170 bp in Vgamma2) and/or the most downstream Jgamma2.3 segment, which can be perceived as 'end-stage' recombinations. Comparative analysis of the TCRG gene configuration in the major immunophenotypic subgroups indicated that TCRgammadelta+ T-ALL display a less mature immunogenotype as compared to TCRalphabeta+ and most CD3- cases. This was reflected by a significantly increased usage of the more downstream Vgamma genes and the upstream Jgamma1 segments. Comparison between adult and pediatric T-ALL patients did not show any obvious differences in TCRG gene configuration. The high frequency, easy detectability, rare oligoclonality, and frequent 'end-stage' recombinations make TCRG gene rearrangements principal targets for PCR-based detection of minimal residual disease (MRD) in T-ALL. We propose a simple heteroduplex PCR strategy, applying five primer combinations, which results in the detection of approximately 95% of all clonal TCRG gene rearrangements in T-ALL. This approach enables identification of at least one TCRG target for MRD monitoring in 95% of patients, and even two targets in 84% of T-ALL.

Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR.

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present in 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde TaqMan-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of < or =10(-4) was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis (95%) are concerned. Furthermore, most IGK-Kde rearrangements (90%) can be used for sensitive detection of MRD (< or =10(-4)) by RQ-PCR analysis.

Fusion gene transcripts and Ig/TCR gene rearrangements are complementary but infrequent targets for PCR-based detection of minimal residual disease in acute myeloid leukemia.

PCR-based monitoring of minimal residual disease (MRD) in acute leukemias can be achieved via detection of fusion gene transcripts of chromosome aberrations or detection of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements. We wished to assess whether both PCR targets are complementary in acute myeloid leukemia (AML). We investigated 105 consecutive AML cases for the presence of fusion gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR): AML1-ETO associated with t(8;21), CBFB-MYH11 with inv(16), PML-RARA with t(15;17), BCR-ABL with t(9;22), and MLL-AF4 with t(4;11). In 17 out of 105 AML cases (16%), fusion gene transcripts were found. Ninety-five of these AML patients (13 with fusion gene transcripts) were also investigated for the presence of IGH, IGK, TCRG and TCRD rearrangements by Southern blot and/or PCR heteroduplex analysis and sequencing. In nine out of 95 patients (9.5%), such rearrangements were found. Combined data revealed that only one patient with a fusion gene transcript had a coexistent Ig/TCR rearrangement. The nine AML patients with Ig/TCR rearrangements, as well as five additional AML patients from a previous study were investigated in more detail, revealing that Ig/TCR rearrangements in AML are immature and unusual. The presence of Ig/TCR rearrangements in AML did not correlate with RAG gene expression levels as determined by real-time quantitative PCR. In conclusion, fusion gene transcripts and Ig/TCR rearrangements are infrequent, but complementary MRD-PCR targets in AML.

Precursor-B-ALL with D(H)-J(H) gene rearrangements have an immature immunogenotype with a high frequency of oligoclonality and hyperdiploidy of chromosome 14.

The IGH gene configuration was investigated in 97 childhood precursor-B-ALL patients at initial diagnosis. Rearrangements were found by Southern blotting in all but three patients (97%) and in 30 cases (31%) we observed oligoclonal IGH gene rearrangements. Heteroduplex PCR analysis revealed at least one clonal PCR product in all Southern blot-positive cases. In 89 patients (92%) complete V(D)J rearrangements were found, while incomplete D(H)-J(H) rearrangements occurred in only 21 patients (22%). In 5% of cases the D(H)-J(H) rearrangements were the sole IGH gene rearrangements. Sequence analysis of the 31 identified incomplete rearrangements revealed preferential usage of segments from the D(H)2, D(H)3 and D(H)7 families (78%). While D(H)2 and D(H)3 gene rearrangements occur frequently in normal B cells and B cell precursors, the relatively frequent usage of D(H)7-27 (19%) in precursor-B-ALL patients is suggestive of leukemic transformation during prenatal lymphopoiesis. Among J(H) gene segments in the incomplete D(H)-J(H) rearrangements, the J(H)6 segment was significantly overrepresented (61%). This observation together with the predominant usage of the most upstream D(H) genes indicates that many of the identified clonal D(H)-J(H) gene rearrangements in precursor-B-ALL probably represent secondary recombinations, having deleted pre-existing D(H)-J(H) joinings. The patients with incomplete D(H)-J(H) gene rearrangements were frequently characterized by hyperdiploid karyotype with additional copies of chromosome 14 and/or by IGH oligoclonality. The presence of incomplete D(H)-J(H) joinings was also significantly associated with a less mature immunogenotype: overrepresentation of V(H)6-1 gene segment usage, absence of biallelic TCRD deletions, and low frequency of TCRG gene rearrangements. This immature immunogenotype of precursor-B-ALL with incomplete IGH gene rearrangements was not associated with more aggressive disease.

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group.

The medium-risk B cell precursor acute lymphoblastic leukemia (ALL) accounts for 50-60% of total childhood ALL and comprises the largest number of relapses still unpredictable with diagnostic criteria. To evaluate the prognostic impact of minimal residual disease (MRD) in this specific group, a case control study was performed in patients classified and treated as medium (or intermediate)-risk according to the criteria of national studies (ALL-BFM 90, DCLSG protocol ALL-8, AIEOP-ALL 91), which includes a good day 7 treatment response. Standardized polymerase chain reaction (PCR) analysis of patient-specific immunoglobulin and T cell receptor gene (TCR) rearrangements were used as targets for semi-quantitative estimation of MRD levels: > or =10(-2), 10(-3), < or =10(-4). Twenty-nine relapsing ALL patients were matched with the same number of controls by using white blood cell count (WBC), age, sex, and time in first complete remission, as matching factors. MRD was evaluated at time-point 1 (end of protocol Ia of induction treatment, ie 6 weeks from diagnosis) and time-point 2 (before consolidation treatment, ie 3 months from diagnosis). MRD-based high risk patients (> or =10(-3) at both time-points) were more frequently present in the relapsed cases than in controls (14 vs 2), while MRD-based low risk patients (MRD negative at both time-points) (1 vs 18) showed the opposite distribution. MRD-based high risk cases experienced a significantly higher relapse rate than all other patients, according to the estimated seven-fold increase in the odds of failure, and a much higher rate than MRD-based low risk patients (OR = 35.7; P= 0.003). Using the Cox model, the prediction of the relapse-free interval at 4 years was 44.7%, 76.4% and 97.7% according to the different MRD categories. MRD-based risk group classification demonstrate their clinical relevance within the medium-risk B cell precursor ALL which account for the largest number of unpredictable relapses, despite the current knowledge about clinical and biological characteristics at diagnosis. Therefore, MRD detection during the first 3 months of follow-up can provide the tools to target more intensive therapy to those patients at true risk of relapse.

Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL.

Sensitive and quantitative detection of minimal residual disease (MRD) in bone marrow (BM) samples of children with acute lymphoblastic leukemia (ALL) is essential for evaluation of early treatment response. In this study, we evaluated whether the traumatic BM samplings can be replaced by peripheral blood (PB) samplings. MRD levels were analyzed in follow-up samples of 62 children with precursor-B-ALL (532 paired BM-PB samples) and 22 children with T-ALL (149 paired BM-PB samples) using real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T cell receptor gene rearrangements with sensitivities of 10(-3) to 10(-5) (one ALL cell in 10(3) to 10(5) normal cells). In 14 of the 22 T-ALL patients, detectable MRD levels were found in 67 paired BM-PB samples: in 47 pairs MRD was detected both in BM and PB, whereas in the remaining pairs very low MRD levels were detected in BM (n = 11) or PB (n = 9) only. The MRD levels in the paired BM-PB samples were very comparable and strongly correlated (r(s) = 0.849). Comparable results were obtained earlier by immunophenotyping in 26 T-ALL patients (321 paired BM-PB samples), which also showed a strong correlation between MRD levels in paired BM and PB samples (r(s) = 0.822). In 39 of the 62 precursor-B-ALL patients, MRD was detected in 107 BM-PB pairs: in 48 pairs MRD was detected in both BM and PB, in 47 pairs MRD was solely detected in BM (at variable levels), and in 12 pairs only the PB sample was MRD-positive at very low levels (

Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes.

Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-based risk group assignment was based on the kinetics of tumor reduction. Consequently, the level of MRD has to be defined precisely in follow-up samples. However, current PCR methods do not allow easy and accurate quantification. We have tested 'real-time' quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle ('real-time') by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the design of patient-specific TaqMan probes at the position of the junctional regions. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-blot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.

Cross-lineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease.

A large series of 202 childhood precursor-B cell acute lymphoblastic leukemia (ALL) patients was analyzed by Southern blotting (SB) for cross-lineage rearrangements and/or deletions in the T cell receptor TCRB, TCRG and TCRD loci. In 93% (187/201) of the precursor-B-ALL patients one or more genes were rearranged and/or deleted. TCRB gene rearrangements were found in 35% (69/196), TCRG gene rearrangements in 59% (113/192), TCRD gene rearrangements in 55% (112/202), and isolated monoallelic or biallelic deletions of TCRD loci in 34% (68/202) of the cases. TCRB gene rearrangements involved exclusively the Jbeta2 locus with complete V(D)Jbeta2 joinings in 53% of gene rearrangements and incomplete Dbeta-Jbeta2 gene rearrangements in 33%. TCRG gene rearrangements frequently occurred on both alleles (65% of cases) and in approximately 70% concerned rearrangements to Jgamma1 gene segments. Most rearranged TCRD alleles (80%) represented incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 gene rearrangements, while the remaining TCRD gene rearrangements remained unidentified. Subsequently, we evaluated, whether heteroduplex PCR analysis of rearranged TCRG and TCRD genes can be used for reliable identification of PCR targets for detection of minimal residual disease (MRD). The concordance between SB and heteroduplex PCR analysis for detection of the various types of clonal TCRG and TCRD gene rearrangements ranged between 78% and 87%. The discrepancies could be assigned to the presence of 'atypical' TCRD gene rearrangements or translocations only detectable by SB, but also to efficient PCR-based detection of rearrangements derived from small subclones, which are difficult to detect with SB. Indications for oligoclonality were observed in 38% and 30% of patients with TCRG and TCRD gene rearrangements, respectively, which is comparable to the frequency of oligoclonality in IGH locus. Based on the combined data it was possible to reduce the broad panel of six TCRD and 12 TCRG primer combinations for MRD studies to two TCRD combinations (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and six TCRG combinations (VgammaI, VgammaII, VgammaIV family-specific primers with Jgamma1.1/2.1 and Jgamma1.3/2.3 primers) resulting in the detection of 80% and 97% of all TCRD and TCRG gene rearrangements, respectively. Finally, the heteroduplex PCR data indicate that MRD monitoring with TCRG and/or TCRD targets is possible in approximately 80% of childhood precursor-B-ALL patients; approximately 55% of patients even have two TCRG and/or TCRD targets.

Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.

It is now widely accepted that the detection of minimal residual disease (MRD) has prognostic value in acute leukemia. However clinical MRD studies need standardized techniques. Therefore, several European laboratories have aligned their goals and performed comparative studies to achieve optimization and standardization of MRD techniques. This was achieved via the BIOMED-1 Concerted Action "Investigation of minimal residual disease in acute leukemia: International standardization and clinical evaluation." This report describes the development of PCR primers and protocols for the detection of MRD in acute lymphoblastic leukemia (ALL) using clone-specific junctional regions of immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. A total of 54 primers was developed (1) to amplify rearrangements of the TCRD, TCRG, and IGK (Kde) genes as well as TAL1 deletions; (2) to sequence the junctional regions and breakpoint fusion regions; and (3) to perform MRD detection in bone marrow or peripheral blood samples during follow-up of ALL patients. Protocols were established to identify PCR targets at diagnosis by performing 25 PCR reactions per patient using appropriate positive and negative controls. Standardized protocols were developed for MRD monitoring via single amplification of the PCR target followed by dot blot hybridization with the corresponding patient-specific junctional region probe. In addition, alternative approaches were designed for cases where the target sensitivity of at least 10(-4) was not obtained. The standardization described here of MRD-PCR techniques is essential for the process of translating MRD research into clinical practice.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vkappa gene family usage in the Vkappa-Kde rearrangements in our series of B-lineage leukemias was comparable to Vkappa gene family usage in functional Vkappa-Jkappa rearrangements in normal and malignant mature B cells, except for a higher frequency of VkappaII family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10(-4) to 10(-5). Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be 'end-stage' rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

The Most Probable Limit of Detection (MPL) for rapid microbiological methods.

Classical microbiological methods have nowadays unacceptably long cycle times. Rapid methods, available on the market for decades, are already applied within the clinical and food industry, but the implementation in pharmaceutical industry is hampered by for instance stringent regulations on validation and comparability with classical methods. Equivalence studies become less relevant when rapid methods are able to detect only one single microorganism. Directly testing this capability is currently impossible due to problems associated with preparing a spiked sample with low microbial counts. To be able to precisely estimate the limit of detection of rapid absence/presence tests, the method of the most probable limit is presented. It is based on three important elements; a relatively precise quantity of microorganisms, a non-serial dilution experiment and a statistical approach. For a set of microorganisms, a limit of detection of one was demonstrated using two different rapid methods.

In vivo properties of a feline herpesvirus type 1 mutant carrying a lacZ insertion at the gI locus of the unique short segment.

The major cause of upper respiratory tract disease in cats in the feline herpesvirus type 1 (FHV-1). FHV-1 replicates predominantly in the mucosal epithelium of the oral and nasal cavities, local immunity should therefore be the key target for vaccine development. Recombinant DNA technology enables accurate manipulation of the genetic content of the FHV-1 genome, hopefully resulting in a next generation of safe vaccine strains that can be used intranasally in cats. Integration of a reporter gene into the glycoprotein I (gI) homologous gene of FHV-1, resulted in strain C4-1-4-1 which displayed reduced replication not only in cell culture but also in the respiratory tract of infected cats. Oronasal application of strain C4-1-4-1 caused less severe clinical signs than local administration of the parent virus. In addition, oronasally vaccinated cats were better protected against the clinical signs of a challenge infection than cats vaccinated subcutaneously.

Vaccination against feline leukaemia using a new feline herpesvirus type 1 vector.

A recombinant feline herpesvirus type 1 (FHV-1) was constructed expressing the envelope glycoprotein gene from feline leukaemia virus (FeLV). The expression cassette containing the long terminal repeat promoter from Rous sarcoma virus was stably integrated at the locus downstream of the gC homologue in FHV-1. Oronasal vaccination with recombinant FHV-1 engendered significant protection against challenge with the homologous FelV-A/Glasgow-1 isolate. Three of four vaccinated cats did not become viraemic for FeLV and developed serum neutralizing antibodies while five of six controls became persistently infected after challenge. However, latent FeLV was detected at 12 weeks after challenge in bone marrow cultures of all animals except one. The potential of this new vector to protect against FeLV was compared with previous reports using live recombinant vaccines.

Transcriptional analysis of the short segment of the feline herpesvirus type 1 genome and insertional mutagenesis of a unique reading frame.

Transcription mapping was performed in the short region of the feline herpesvirus type 1 (FHV-1) genome as a first approach to the functional analysis of open reading frames encoding the homologs of the herpes simplex virus type 1 (HSV-1) gD, gl, gE, US9, and probably also US8.5. All reading frames appeared to be transcribed. Transcripts were grouped into two nested RNA sets; namely, the coterminal transcripts of gD and gl and the coterminal transcripts of gE, US8.5, and US9. This situation was similar to that found in the equivalent region of HSV-1. The FHV-1 ORFs US8.5 and US9 have not been described previously. Sequence analysis and comparison of the putative polypeptide encoded by US8.5 revealed that this ORF was unique to FHV-1. However, US8.5 of FHV-1 might be functionally related to its positional homologs in HSV-1 and equine herpesvirus type 1. In all three viruses, US8.5 does not seem to be critical for virus propagation in cell culture. This was shown for FHV-1 by isolating a mutant containing an insertion in US8.5 and comparing its growth properties in cell culture to those of the parent virus G2620. With regard to US9, conscientious amino acid sequence alignment of FHV-1 US9 and homologs in related herpesviruses suggests that this particular protein could contribute to the virus infectivity in vivo. This speculation was based on the highly conserved C-terminus of US9, starting with a characteristic YYSES motif and followed by a nuclear target sequence and a transmembrane region.

The gene downstream of the gC homologue in feline herpes virus type 1 is involved in the expression of virulence.

Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a beta-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 21K. RNA analysis indicated a corresponding transcript of 0.8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.