mdv1-miR-M7-5p, located in the newly identified first intron of the latency-associated transcript of Marek's disease virus, targets the immediate-early genes ICP4 and ICP27.

Marek's disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5' end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3' end and allowed identification of the 5' end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3' UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

Kinetic expression analysis of the cluster mdv1-mir-M9-M4, genes meq and vIL-8 differs between the lytic and latent phases of Marek's disease virus infection.

Marek's disease virus (GaHV-2) is an alphaherpesvirus that induces T-cell lymphoma in chickens. The infection includes both lytic and latent stages. GaHV-2 encodes three clusters of microRNAs (miRNAs) located in the internal (I)/terminal (T) repeat (R) regions. We characterized transcripts encompassing the mdv1-mir-M9-M4 and mir-M11-M1 clusters located in the IR(L)/TR(L) region, upstream and downstream from the meq oncogene, respectively. By 5'- and 3'-RACE-PCR and targeted RT-PCR, we showed that mdv1-mir-M9-M4 could be transcribed from an unspliced transcript or from at least 15 alternatively spliced transcripts covering the IR(L)/TR(L) region, encompassing the meq and vIL-8 genes and localizing the mdv1-mir-M9-M4 cluster to the first intron at the 5'-end. However, all these transcripts, whether spliced or unspliced, seemed to start at the same transcriptional start site, their transcription being driven by a single promoter, prmiRM9M4. We demonstrated alternative promoter usage for the meq and vIL-8 genes, depending on the phase of GaHV-2 infection. During the latent phase, the prmiRM9M4 promoter drove transcription of the meq and vIL-8 genes and the mdv1-mir-M9-M4 cluster in the first intron of the corresponding transcripts. By contrast, during the lytic phase, this promoter drove the transcription only of the mdv1-mir-M9-M4 cluster to generate unspliced mRNA, the meq and vIL-8 genes being transcribed principally from their own promoters. Despite the expression of meq and the mdv1-mir-M9-M4 cluster under two different transcriptional processes during the latent and lytic phases, our data provide an explanation for meq expression and mdv1-mir-M4-5P overexpression in miRNA libraries from GaHV-2-infected cells, regardless of the phase of infection.

ICP27 protein of Marek's disease virus interacts with SR proteins and inhibits the splicing of cellular telomerase chTERT and viral vIL8 transcripts.

All herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Marek's disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Marek's disease.

Sequential autoprocessing of Marek's disease herpesvirus protease differs from that of other herpesviruses.

Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)(P3)-X(P2)-A(P1)/S(P1') (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M(P3)-N(P2)-A(P1)/S(P1'). Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

Major rearrangements in the E element and minor variations in the U3 sequences of the avian leukosis subgroup J provirus isolated from field myelocytomatosis.

We collected paraffin-embedded myelocytomatoses induced by subgroup J avian leukosis virus (ALV-J) in French poultry from 1992 to 2000. We used nested PCR to obtain the U3 LTR and the E element sequences that encompass putative binding sites for transcription factors. We observed minor mutations in the U3 sequences that rarely affected transcription factor binding sites, thus preserving the transcriptional potential of the U3 LTR. However, we observed a large variability in the E element sequences from both field and experimental tumor samples. This variability involved genomic rearrangements and various deletions that most often occurred between two direct repeat sequences. Moreover, in seven DNA samples of the 22 field tumors analyzed, we observed two different sequences for the E element region, suggesting that proviral genomes of two different sizes may be simultaneously present in a tumor. Even though most of the E element sequences were mutated or rearranged, all myelocytomatosis samples always exhibited one E element sequence containing at least one putative C/EBP binding site that was unaffected and still potentially functional.

Molecular epidemiology of European brown hare syndrome virus in France between 1989 and 2003.

Genetic diversity between French European brown hare syndrome (EBHS) viruses since the disease appeared has been evaluated. Nucleotide sequencing of the partial capsid protein genes of 169 EBHS viruses collected from various parts of France between 1989 and 2003, three reference strains, and a Greek EBHSV collected in 2002 revealed a maximum nucleotide divergence of 11.7%, indicating a high level of conservation between viruses. Two major groups were identified. The first group contained EBHS viruses collected since 1989 from different parts of France, the reference strains, and all of the viruses located in the far north of France. In this group, three genogroups were clearly identified as mainly related to their geographic origin. The distribution of the viruses suggests that the early viruses have not disappeared and have slowly evolved in their area of origin. The second group, supported by a significant bootstrap value, contained the Greek EBHSV with the French EBHS viruses collected between 1999 and 2003 from regions of southern France. It constitutes a newly identified genogroup. Our results demonstrate strong differences in genetic evolution between EBHSV and rabbit haemorrhagic disease virus, with persistence of the earlier EBHS viruses and interaction between the geographical and temporal distributions.

[Viruses and telomerase]. / Virus et télomérase.

The involvement of viral infections in the genesis of cancers is currently established. However, the narrow correlation between viruses and the activation of telomerase was defined only recently. The telomerase activity is detected in more than 85 % of human cancers and would take part to immortalization processes, associated with cellular transformation. The active minimal complex of telomerase is composed of a RNA subunit comprising a template sequence which is reverse-transcribed onto telomeres by a proteic component TERT. A disregulation of telomerase activity was observed consecutively to infection by viruses from various families. This phenomenon would mainly rest on modulation of the expression and the nuclear targeting of TERT by viral proteins or further to viral genome integrations close to the TERT gene. Moreover, the Marek's Disease herpesvirus, responsible of T-lymphomas in chickens, is particular in that it encodes, within its genome, a viral telomerase RNA subunit, vTR, which induces a more greater telomerase activity than its avian cellular counterpart, cTR.

Induction of telomerase activity in avian lymphoblastoid cell line transformed by Marek's disease virus, MDCC-MSB1.

Telomerase has been studied extensively in human and murine tumors, but little is known about the role of telomerase in the tumor biology of other vertebrate species such as the chicken. We studied the telomerase activity of the lymphoblastoid cell line derived from lymphomas induced by Marek's disease virus (MDCC-MSB1) compared with another avian cell line (PA5) and peripheral blood lymphocytes (PBL) using the telomeric repeat amplification protocol (TRAP) Assay. Telomerase activity in MDCC-MSB1 was 4.5 times greater than in the PA5 cell line and normal avian lymphocytes. These results demonstrate for the first time that telomerase is more intense in one transformed cell line than in normal cells, suggesting a potential role for telomerase in carcinogenesis induced by an avian virus.

Single-nucleotide polymorphisms in two Marek's disease virus genes (Meq and gD): application to a retrospective molecular epidemiology study (1982-1999) in France.

Marek's disease virus (MDV) is a herpesvirus that causes a lymphoproliferative disease in chickens. Vaccines against MDV are available, but the virus is gradually becoming more virulent. A molecular epidemiology study of MDV was carried out by assessing nucleotide variation in two different genes, Meq and gD, in 68 French field isolates circulating from 1982 to 1999, compared with reference strains. Viral DNA was amplified by nested PCR and sequenced directly. Comparison of the nucleotide sequences revealed a high nucleotide sequence identity (98 %). Single-nucleotide polymorphisms were identified, leading to the identification of three gene alleles for gD and six for Meq. Nine combinations of alleles were identified. A majority of French isolates (60.5 %) clustered in the C1 type, which has been present for over 17 years. Waves of non-C1-type isolates appeared when vaccine efficacy decreased. Furthermore, specific discriminating sequences were obtained for the CVI-988 vaccine strain.

Assembly of Marek's disease virus (MDV) capsids using recombinant baculoviruses expressing MDV capsid proteins.

The genes UL18, UL19, UL26, UL26.5, UL35 and UL38 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the homologues of herpes simplex virus type 1 (HSV-1) capsid proteins VP23, VP5, VP21-VP24, preVP22a, VP26 and VP19C, were identified and sequenced. Recombinant baculoviruses were used to express the six capsid genes in insect cells. Coexpression of the six genes or of UL18, UL19, UL26.5 and UL38 in insect cells resulted in the formation of capsids with a large core. In addition, electron microscopy of thin sections clearly revealed the presence of large numbers of small spherical particles. Experimental coinfection demonstrated that these small particles were associated with production of the preVP22a protein.

Evolution of preproinsulin gene in birds.

The coding region of the preproinsulin gene has been cloned and partly sequenced in a variety of marine and terrestrial birds (28 species). All genes showed the "ancestral" structure with a large intron-2. The size of intron-2 changed considerably during the evolution of birds (2.4-4.2kb). The hydrophobicity of signal peptides was conserved. Bird C-peptides were predicted to be 28 amino acids long, but circulating C-peptides would be only 26 amino acids long, with Passer as a possible exception. Bird C-peptides were found to lack the sequences identified in mammals as responsible for peptide bioactivity and the structure of the central part. In contrast, predicted insulin sequences were highly conserved. Only two types of analog were identified: the hypoactive form (GluA8), present only in Anseriformes and the hyperactive form (His A8), present in all other species. Based on 3'-nucleotide sequence analysis (extending into intron-2), birds appeared to be monophyletic. Five groups were clearly identified: Paleognathae, Galliformes, Anseriformes, Passeriformes, and Charadriiformes. Paleognathae were suggested as the basal group, supporting the traditional view of avian evolution. Subsequent branching identified a gallo-anserae group and a group containing all other Neognathae. Surprisingly, Columba livia (Columbiforme order) clustered with Galliformes. With represented species, Procellariiformes and possibly Ciconiiformes, and Pelicaniformes were suggested as paraphyletic, in agreement with conclusions from some studies based on mitochondrial DNA sequences.

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants.

The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant.

Folding of the rabbit hemorrhagic disease virus capsid protein and delineation of N-terminal domains dispensable for assembly.

Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are caliciviruses that produce severe symptoms and are lethal to rabbits and hares. The folding of the capsid protein was studied by determination of the antigenic pattern of chimeric capsid proteins, composed of regions from RHDV and EBHSV capsid proteins. The anti-RHDV monoclonal antibody (MAb) E3, which is known to bind an external conformational epitope, recognized the RHDV C-terminal region. The anti-RHDV MAb A47, which binds a buried epitope, recognized the RHDV N-terminal region. Using a pGEX expression library, we more precisely mapped the MAb A47 epitope on a 31 residues length peptide, between residue 129 and 160 of the VP60, confirming its location in the N-terminal part of the protein. These results demonstrate that the C-terminal part of the protein is accessible to the exterior whereas the N-terminal domain of the protein constitutes the internal shell domain of the particle. With the aim of using virus-like particles (VLPs) of RHDV as epitope carriers or DNA transfer vectors, we produced in the baculovirus system three proteins, DeltaN1, DeltaN2 and DeltaN3, truncated at the N terminus. The DeltaN1 protein assembled into VLPs, demonstrating that the first 42 amino acid residues are not essential for capsid assembly. In contrast, DeltaN2, from which the first 75 residues were missing, was unable to form VLPs. The small particles obtained with the DeltaN3 protein lacking residues 31 to 93, located in the immunodominant region of the RHDV capsid protein, indicate that up to 62 amino acid residues can be eliminated without preventing assembly.

Detection of avian oncogenic Marek's disease herpesvirus DNA in human sera.

The avian herpesvirus Marek's disease virus (MDV) has a worldwide distribution and is responsible for T-lymphoma in chickens. The question as to whether MDV poses a public health hazard to humans was first raised when the virus was isolated in 1967. However, no irrefutable results have been obtained in immunological and virological studies. We used a nested-PCR to detect MDV DNA in human serum samples. A total of 202 serum samples from individuals exposed and not exposed to poultry was tested by nested-PCR for a target sequence located in the MDV gD gene. The assay system was specific and sensitive, making it possible to detect a single copy of the target sequence. Forty-one (20%) of the 202 serum samples tested positive for MDV DNA. The prevalence of MDV DNA was not significantly different in the group exposed to poultry and the group not exposed to poultry. There was also no difference due to age or sex. Alignment of the 41 gD sequences amplified from human sera with eight gD sequences amplified from MDV-infected chicken sera showed a maximum nucleotide divergence of 1.65%. However, four 'hot-spot' mutation sites were identified, defining four groups. Interestingly, two groups contained only human MDV-gD sequences. The status of the MDV genome detected in human blood is discussed.

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16.

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.

Identification of a new cleavage site of the 3C-like protease of rabbit haemorrhagic disease virus.

The calicivirus rabbit haemorrhagic disease virus (RHDV) possesses a 3C-like protease which processes the RHDV polyprotein. In order to monitor the proteolytic activity of the RHDV 3C-like protease on its putative target sequences, i.e. the 10 EG dipeptide bonds distributed along the large polyprotein, a new approach was carried out. Preliminary experiments showed that the luciferase gene when fused in-frame with a long gene yielded a fusion protein almost devoid of luciferase activity. This reporter system was used to test which EG dipeptide bonds were cleaved by the RHDV protease when the coding sequences of the dipeptides and their flanking sequences were inserted at the junction between the protease and luciferase genes. The coding sequences of the fusion proteins were cloned downstream of the T7 promoter and the proteolytic activity was evaluated by measuring the luciferase activity in both in vitro and 'in vivo' systems. The EG dipeptide bonds at positions 718-719, 1108-1109 and 1767-1768 were confirmed as cleavage sites and a new cleavage site EG (143-144) was identified.

Molecular epidemiology of rabbit haemorrhagic disease virus outbreaks in France during 1988 to 1995.

In order to evaluate genetic variation between rabbit haemorrhagic disease virus (RHDV) isolates and to derive phylogenetic relationships, 56 virus isolates collected from various parts of France over a 7 year period (1988 to 1995) were examined. Analyses were carried out by direct nucleotide sequencing of PCR fragments of three genomic regions encoding the capsid protein (VP60) (regions A and B) and a non-structural protein (region C). Multiple sequence alignments revealed maximum nucleotide divergence of 7.6, 9.4 and 8.7% for regions A, B and C, respectively, indicating a high level of conservation between isolates. Irrespective of the genomic region analysed, phylogenetic analyses carried out using various methods allowed the identification of three genogroups; distribution of isolates within these genogroups appears to be more related to the year of their collection than to their geographical origin. The possible evolution of RHDV is discussed.

Structural, antigenic and immunogenic relationships between European brown hare syndrome virus and rabbit haemorrhagic disease virus.

The capsid protein of a French isolate of the European brown hare syndrome virus (EBHSV) was expressed in the baculovirus system. The recombinant EBHSV (rEBHSV) capsid protein was able to self-assemble into virus-like particles (VLPs). The VLPs were indistinguishable from the infectious EBHSV and displayed morphological characteristics similar to those we have described for the VLPs resulting from the expression of the capsid protein of rabbit haemorrhagic disease virus (RHDV), a closely related calicivirus. Cross-protection experiments showed that vaccination with rEBHSV particles did not protect rabbits against an RHDV challenge. A set of monoclonal antibodies (MAbs) was raised against rEBHSV capsid protein and used together with anti-RHDV and anti-EBHSV MAbs produced against native viruses to study the antigenic relationships between the two caliciviruses. All six anti-EBHSV MAbs delineated discontinuous epitopes; two of them reacted with specific surface epitopes and the remaining four reacted with internal epitopes which were also present in rRHDV. All anti-RHDV MAbs were monospecific; three reacted with surface linear epitope(s), two reacted with internal linear epitope(s) and one with a surface conformational epitope. On the basis of all these results, a classification of RHDV and EBHSV as two serotypes of a single serogroup is proposed.

Bivalent binding of a neutralising antibody to a calicivirus involves the torsional flexibility of the antibody hinge.

The structure of a complex between rabbit haemorrhagic disease virus (RHDV) virus-like particles (VLPs) and a neutralising monoclonal antibody mAb-E3 has been determined at low resolution by cryo-electron microscopy and three-dimensional (3-D) reconstruction techniques. The atomic co-ordinates of an Fab were fitted to the cryo-electron microscope density map to produce a binding model. The VLP has a T = 3 icosahedral lattice consisting of a hollow spherical shell with 90 protruding arches. Each dimeric arch presents two mAb binding sites; however, steric hindrance between the variable domains of the Fabs prevents the occupation of both sites simultaneously. Thus the maximum mAb occupation is 50%. Once a mAb is bound to one site it may bind to either of two neighbouring sites related by a local 3-fold axis. The mAbs are bound bivalently on epitopes not related by a 2-fold symmetry axis. This binding geometry implies a torsional flexibility of the mAb hinge region, involving a 60 degrees rotation of one Fab arm with respect to the other. Owing to extreme flexibility of the hinge region, the Fc domains occupy random orientations and are not visible in the reconstruction. The bivalent attachment of mAb-E3 to RHDV suggests that the neutralisation mechanism(s) involves inhibition of viral decapsidation and/or the inhibition of binding to the receptor.

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.