[Severe pulmonary embolism revealed by status epilepticus]. / Embolie pulmonaire révélée par un état de mal épileptique.

INTRODUCTION: High-risk pulmonary embolism (PE) is associated with high mortality rate (>50%). In some cases, diagnosis of PE remains a challenge with atypical presentations like in this case report with a PE revealed by status epilepticus. CASE REPORT: We report the case of a 40-year-old man without prior disease, hospitalized in ICU for status epilepticus. All paraclinical examinations at admission did not show any significant abnormalities (laboratory tests, cardiologic and neurological investigations). On day 1, he presented a sudden circulatory collapse and echocardiography showed right intra-auricular thrombus. He was treated by thrombolysis and arteriovenous extracorporeal membrane oxygenation. After stabilization, computed tomography showed severe bilateral PE. He developed multi-organ failure and died 4days after admission. CONCLUSIONS: Pulmonary embolism revealed by status epilepticus has rarely been reported and is associated with poor prognosis. Physicians should be aware and think of the possibility of PE in patients with status epilepticus without any history or risk factors of seizure and normal neurological investigations.

Oligomerization paths of the nucleoprotein of influenza A virus.

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.

Replication and packaging of an infectious bursal disease virus segment A-derived minigenome.

A synthetic cytomegalovirus (CMV) promoter-driven cDNA minigenome containing the enhanced green fluorescence protein (EGFP) gene as a reporter was derived from the genomic segment A of infectious bursal disease virus (IBDV). The 5'-end of the minigenome was fused to the transcription start site of the immediate early CMV promoter, and the hepatitis delta virus (HDV) ribozyme sequence was added at its 3'-end. We show that co-transfection of the minigenome with a plasmid encoding the IBDV RNA-dependent RNA polymerase VP1, results in a consistent increase of the EGFP expression, as measured by fluorescence microscopy and flow cytometry assays. Replication of the minigenome-derived transcript was evidenced by real-time RT-PCR analyses targeted to both the plus- and minus-sense strands. When cells were infected with IBDV and transfected with the plasmid carrying the minigenome, the minigenome was packaged and EGFP was found to be expressed in a second cycle of infection. These results show the potential use of this system as a new tool to characterize IBDV replication and genome packaging.

[Schizophrenia diagnostic announcement in a French psychiatric unit]. / Annonce du diagnostic de schizophrénie au sein d'un service de psychiatrie de secteur.

Announcement of schizophrenia diagnostic to the patients is a topical issue in France. The evolution in clinical practices, a better efficiency in therapeutic procedures and the fundamental right of the patient to obtain information have initialised the discussion of its interest. Spontaneous claim for information from the patient is rarely observed although awareness troubles might be reported at the instauration of the mental disorder or during its evolution. Methodological studies concerning the diagnostic announcement are limited. Except the Bayle studies recently published, only a few publications are available in France about the knowledge of their pathology and their need to be clearly informed. French scientific literature deals generally about medico-legal aspects of this information and consisted of survey about diagnostic announcement. International literature is more abundant and presents positive and negative aspects of the announcement. An information procedure of schizophrenia announcement to the patient has been developed in our hospitalisation unit of psychiatry. This procedure has taken place on the basis of the literature data, our specificity and our clinical experiences. For some Anglo-American psychiatrists who have proceeded to semi-structured interview in order to announce the diagnostic, information to the patients might improve the clinical relationship. Thus, compliance to the treatment is significantly increased. The ability of the patient to recognise the symptoms of the disease and to accept their consequences and the treatments is associated to a better social prognosis, daily activities and response to the treatment. The announcement impact justifies the prescription of neuroleptics, treatment that is notoriously perceived as prejudicial by the patients themselves or more commonly in the basic population. To obtain compliance to the treatment, a satisfactory acceptance of the mental disorder is required. Compliance is based on satisfactory information in order to gain the cooperation of the patient and its relative (10). Atkinson has classified four main types of arguments, the ethical principle to be informed, talk to explain and give sense to the symptoms, reduce the feeling of guilt perceived by the patient and his relative and enhance the collaboration between the patient and the nursing staff. According to Ferreri and Bayle studies French psychiatrists reluctance to announce schizophrenia diagnostic are the following: lack of request or of interrogations asked by the patient about their disease, diagnostic and prognosis uncertainty and irreversibility of the disease, complexity of the pathology and its origin which hinder an accessible explanation, cognitive disorders frequently observed with schizophrenic patients which may be associated with difficulties of understanding information, destabilization of the patient-nursing staff relationship and social stigmatisation risks. Other arguments like reluctance to give a "label" to the disease, too abstract diagnostic, a negative social vision and the possibility of discouragement for the relative are classically retrieved in French literature. In fact, divulgation of the term schizophrenia involves a panel of negative representations and is hindered by the confusion in the social imagination of such a term related with lost of control, quintessence of madness, dangerous behaviour possibilities, evil and incurability. Some psychiatrists do not transmit information arguing that significant obstruction of the future may be consecutive to the information. They prefer to use vague terms more socially acceptable like "nervous breakdown or depression, atypical or emotional disorder, dissociative troubles...". Information to the patient about his mental disorder is more frequent in psychiatry for affective, anxious and additive troubles than for schizophrenia. Our procedure of diagnostic announcement has been elaborated after preliminary discussion with the medical and nursing staff. Diagnostic of schizophrenia announcement has been presented by weighing the pros and cons according to the intemational literature. It clearly appeared that benefits for the patients prevail on the drawbacks. Nevertheless, inclusion and clinical supervision have to be carefully precised in particular to verify the ability to receive information. Short term objectives: deliver progressively information to the patient about his disease by means of an active and educational process with hope and optimism using a accessible language (explanation of each terms used with the intention of being well understood); quantify the impact of diagnostic announcement on the schizophrenic patient using clinical rating scales during a period of one month (clinical interview at day 1, day 7 and day 28). Mid term objectives: improve the global supervision and autonomy of schizophrenic by means of a therapeutic project helping the patient to become an active partner in the monitoring of his mental disorder; enhance a psycho-educational program after the procedure of announcement in order to optimise the observance of his treatment, increase his quality of life and answer to the requests of his relative; 45 patients (age 29.3 +/- 8.8 years old) have been included to be informed on their diagnostic since the elaboration of this procedure during a time period of 24 months. Time interval between the beginning of their pathology and the delivering of this information was 4.7 years. Most of them (56%) presented a paranoid type of schizophrenia. In most of the cases, the patients did not know their diagnostic or declared suffering from a diagnostic, which was erroneous; 80% of the 45 patients have complied with the procedure until its end. On more than 24 of following after the instauration of the diagnostic announcement procedure, these patients ha ve presented satisfactory observance to the medical supervision (medical consultation and drug intake); 60% of the patients were regularly present to their medical appointment. The number of patients included (45 patients) appears small compared to the time interval of the study (24 months) but was significant according to the great changes in our clinical approach. Thus, this procedure was not systematically applied, in particular the patients who did not want to be informed on their disease. Is it clinically relevant or not to announce diagnostic of schizophrenia to the patient? This issue remains questioned according to the few studies published at the present time, any consensus has been clearly presented on formal indications or contra-indications. If on an ethical side, this information appears logical, the medical and nursing staff should require special care. Special care must be taken before delivering information to the patients; each situation must be evaluated in order not to comply with an ideology of total and inadequate information, which could have serious consequences. Nevertheless, it appeared clearly that information must be given to stabilized patients with satisfactory insight. Moreover, psychotherapeutic projects become easier because patients awareness and understanding towards pathological symptoms are greatly improved. Partnership between patient and medical staff is the key of this dynamic and psycho-educative procedure, which opens new horizons in our therapeutic prospect.

Long-term assessment of neuropsychiatric adverse reactions associated with efavirenz.

OBJECTIVES: The Sensio study objectives were to assess the outcome of neuropsychiatric adverse reactions (NPAR) that develop after initiation of efavirenz (EFV) therapy, to ascertain the late NPAR after a 3-month treatment period, to evaluate the impact of NPAR on patients' quality of life (QoL) in a real-life population. METHODS: During a 6-month period, consecutive HIV-infected adult outpatients receiving an ongoing EFV therapy for at least 3 months were asked to fill in a specifically designed self-administered questionnaire addressing sleep disturbances, behavioural changes, mood disturbances, anxiety, cognitive disorders, hallucinations, dizziness and the general impact on patients' QoL. RESULTS: A total of 174 questionnaires were analyzed. The main late emergent NPAR were sleep disorders: abnormal dreams 24.7%, nocturnal waking 19.6%, trouble falling asleep 17.8%; cognitive disorders: memory disorders 23.0%, impaired concentration 18.9%; anxiety 15.5%; mood disorders: sadness 19.3%, suicidal ideations 9.2%. Global neuropsychic discomfort was moderate to severe in 23% of patients after a 3-month treatment period. CONCLUSION: NPAR occur mainly during the first month of EFV therapy but often persist thereafter. A significant percentage of patients reported suicidal ideations at the time of the study. Our results suggest the need for routine screening for NPAR among patients receiving EFV therapy and better management.

[Hepatic tolerance of atypical antipsychotic drugs]. / Tolérance hépatique des antipsychotiques atypiques.

The strategy in the choice of antipsychotic agent must take into account the hepatic tolerance according to non-negligible incidence of liver disorders among psychiatric population (presence of risk factors like alcoholism, drugs of abuse intake, polymedication including potentially hepatotoxic drugs.). More than 1 000 drugs have been listed as being responsible of hepatic side effects; 16% of these agents were neuropsychiatric drugs. Antidepressive drugs (tricyclic agents or SSRI), mood stabilizing agents and neuroleptic drugs have been implicated in biological or/and clinical hepatotoxicity. For these reasons, some psychotropic agents have been withdrawn of the pharmaceutical market like alpidem or medifoxamine. Atrium*, sometimes used to correct tremor induced by neuroleptic drugs, has been withdrawn recently, as well. Isolated elevations of hepatic enzymes occur frequently with phenothiazines drugs (frequency evaluated to 20%) but also with other classes of neuroleptic agents, as well. On the contrary, clinical hepatitis have been more rarely described with neuroleptic drugs like phenothiazine agents (0,1-1%) or with haloperidol (0,002%). The definition of hepatotoxicity is based on biological parameters (elevation of alkaline phosphatase enzyme, SGPT, SGOT and GGT) or on clinical abnormalities (hepatitis, jaundice.). Clinical hepatitis could be either cytolytic or cholestatic. Clinical diagnosis and the research of its origin may include many investigations like abdominal ultrasonogram and percutaneous liver biopsy. The present article describes the cases of hepatic disorders reported with AAD (Atypical Antipsychotic Drugs), which are available in France (amisulpride, clozapine, olanzapine, risperidone). This new pharmacological class of antipsychotic drugs has showed great interest to improve negative symptoms of schizophrenia and to reduce disabling side effects like dystonia. According to the bibliographic data available, the following points and information must be clinically taken into account. Frequency of hepatic troubles: according to the bibliographic data, AAD appeared generally well tolerated in most cases. The frequency of hepatic troubles remains in general very low or rare. The cases published were observed with clozapine, olanzapine and risperidone. Nevertheless, some authors have observed higher frequency of hepatic enzymes elevation with some AAD. In an investigation comparing hepatic tolerance of clozapine (n=167) versus haloperidol (n=71), 37,3% of clozapine treated patients showed a relevant SGPT increase versus 16,6% with haloperidol. Nature of the hepatic troubles: among the clinical observations, asymptomatic biological disorders of the hepatic function are generally described but cytolytic or cholestatic hepatitis were reported, as well. Symptomatic hepatic dysfunctions were, sometimes, associated with other disorders like convulsions, pneumonia or malignant syndrome. Thus, hepatic check-up may be relevant in case of significant side-effect outcome. Delay time before the hepatic episode: hepatic injuries generally occurred within the first weeks of treatment but this delay highly varied in the literature from 1 to 8 weeks, 12 days to 5 months, 1 day to 17 months for clozapine, olanzapine and risperidone, respectively. These delay times are very similar to those observed with other psychotropic drugs. Reversibility of the hepatic troubles and rechallenge of the responsible agent: all cases were reversible after the AAD withdrawal except with one patient (39 years old) treated by clozapine (350 mg/day) who developed a fulminant and irreversible hepatitis after 8 weeks of monotherapy. In most cases, the AAD was withdrawn after the hepatic episode according to the significant risk of irreversible alteration. Nevertheless, normalization of hepatic enzymes has been described despite AAD maintenance at the same dosage or after dosage reduction. Rechallenge of clozapine after a first episode was performed for three patients, only one redeveloped a new hepatic disorder. According to different authors, special care is required if maintenance or rechallenge of the agent is indispensable after a first episode of isolated hepatic enzyme elevation (i.e resistance or intolerance to other treatments). In this case, biological and clinical supervision has to be carefully scheduled, which demands a satisfactory compliance from the patient. On the contrary, in case of clinical hepatotoxicity, rechallenge or maintenance is absolutely inadvisable. Mechanism of the hepatic troubles: precise mechanisms of the hepatotoxicity remain unclear. Contrary to phenothiazine drugs, no information is available on the respective rule of the agents and their metabolites. Hypersensitivity syndrome or eosinophilia has been reported, suggesting a possible immuno-allergic mechanism. Presence of risk factors: risk factors have been retrieved, in some observations, like high daily dosage, high plasmatic concentration, age, alcoholism, obesity or antecedent of hepatic disorders like Gilbert syndrome. Special care is advisable with these patients. As hepatotoxicity has been observed after surdosage (or suicide attempt), a hepatic check-up has to be performed in these clinical situations. Co-medication with hepatotoxic drugs may increase the risk as it has been suggested. In many observations, co-medication made difficult the incrimination of the AAD in the hepatic disorders outcome. Monotherapy has the great advantage to make easier the withdrawal of the responsible agent and its substitution. As drugs of abuse like cocaine or ecstasy are notoriously responsible of hepatotoxicity, they represent a probable factor of risk. Moreover, their detection is fundamental during the clinical investigation. Conclusion - Diagnosis of toxic hepatitis is mainly based on the chronology between agent introduction and hepatic disorder onset but other causes must be excluded. Bibliographic data analysis greatly contributes to confirm toxic hepatitis diagnosis. Nevertheless, this article emphasized the limits of bibliographic review to compare drugs towards tolerance. Most of the bibliographic data were case-reports for which it was sometimes difficult to provide absolute evidence of the responsibility of the agent. Moreover, spontaneous notification to health national administration is rarely systematic, in particular with isolated elevation of hepatic enzymes, and even more rarely published in international reviews. Nevertheless, according to the present data available in the literature, systematic and regular hepatic survey does not seem necessary in absence of risk factors. As for other side effects, which may occur more or less rapidly, great advantages may be obtained from psycho-education programs associating the patients in order to detect the first symptoms. Because little long-term hepatic follow-up comparing AAD is available, controlled studies should be carried out to precise the frequency and the risk factors (covariables) to prevent hepatitis outcome.

Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout.

The causative viruses of two diseases of rainbow trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN), exert much of their cytopathogenic effect in cell culture through the induction of apoptosis. In the present study, the TUNEL procedure was used to investigate the presence of apoptotic cells in different organs of rainbow trout infected with the viruses of VHS and IPN. VHS viral infection resulted in massive apoptosis in renal lymphoid tissue, where viral antigens were also detected. Large numbers of viral particles were observed in close proximity to apoptotic cells. Apoptosis was not detected in excretory cells of the renal tubules or in infected muscle cells. IPN virus did not induce apoptosis in the pancreas. However, the DNA degradation associated with apoptotic nuclei was observed in muscle lesions. Taken together, these results indicated that induction of apoptosis in vivo was critically influenced by the species of virus and the cell type. Moreover, it would seem likely that apoptosis contributed to the nature of the two diseases and to mortality.

Construction of avian adenovirus CELO recombinants in cosmids.

The avian adenovirus CELO is a promising vector for gene transfer applications. In order to study this potentiality, we developed an improved method for construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method, the ability to produce infectious particles and the stability of the genome were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). Our aim was to develop a replication-competent vector for vaccination of chickens, so we first generated knockout point mutations into 16 of the 22 unassigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs. An expression cassette coding for the enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then retained the vector backbone with the largest deletion (i.e., 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease virus proteins. These CELO vectors could be useful for vaccination in the chicken species.

Real-time quantitation of bcr-abl transcripts in haematological malignancies.

We have applied an automated real-time quantitative PCR assay using a double-labeled fluorogenic probe to detect t(9;22)-positive cells in haematological malignancies. The results are expressed as the ratio of chimeric bcr-abl transcripts on abl transcripts. Highly reproducible results were obtained for t(9;22)-positive K562 RNA. Ten copies of bcr-abl DNA from a recombinant KW-3 plasmid and one positive cell in 10(4) can be detected. Thirty-two patients with chronic myeloid leukaemia (CML), 25 with acute leukaemia, 12 with myelodysplastic syndromes and 7 with other myeloproliferative syndromes were tested. Follow-up data were obtained in bcr-abl positive cases. Results were compared with those of conventional nested RT-PCR and cytogenetics. Real-time quantitative RT-PCR values correlated well with both these methods. However, in some cases the only means of detecting early relapse or blastic transformation was to examine the kinetics of real-time quantitative RT-PCR. Thus, real-time quantitative RT-PCR appears suitable for the diagnosis and follow-up of patients with the t(9;22) translocation.

Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites.

The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine-lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)-X-Ala downward arrowAla motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis- and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease.

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus.

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

Fish rhabdovirus cell entry is mediated by fibronectin.

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.

Obtention of porcine aminopeptidase-n transgenic mice and analysis of their susceptibility to transmissible gastroenteritis virus.

To obtain a laboratory animal model for transmissible gastroenteritis virus (TGEV) infection, transgenic mice (Tg) were produced by introducing two porcine aminopeptidase-n (APN) cDNA-derived constructs into the mouse genome. In the first construct, the APN cDNA was fused in 5' with the 1 kb upstream region of the APN gene and in 3' with the SV40 small intron and polyadenylation site. In the second construct, the 5' end of the APN cDNA was replaced by the corresponding domain of the APN gene comprising the three first introns, an additional intron (the rabbit beta-like globine intron 2) was inserted at the 3' extremity of the construct and the resulting DNA stretch was placed under the control of the rat intestinal fatty acid-binding protein (I-FABP) gene promoter. Transgenes were obtained with these two constructs, and RNA expression was evidenced by RT-PCR with the second construct in a transgene lineage. Using two different immunoassays, expression of the porcine APN protein was not detected in the transgenic intestines of animals of the RT-PCR positive lineage. Northern blot analyses did not revealed TGEV replication in infected adult mice. Additional assays will be carried out on young animals to detect potential TGEV susceptibility.

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment.

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

[Once-daily administration of didanosine in combination with anti-retroviral zidovudine in previously untreated patients]. / Administration en prise unique quotidienne de la didanosine dans une association anti-rétrovirale avec la zidovudine chez des patients non préalablement traités.

25 HIV-infected antiretroviral-naive adults were included in a 24-week study to evaluate the efficacy and the tolerability of a zidovudine/didanosine combination therapy in which didanosine was administered once daily (200 mg if weight < 60 kg, 300 mg if weight > 60 kg) and zidovudine twice daily (500 mg/day if weight < 90 kg, 600 mg/day if weight > 90 kg). 5 patients discontinued their treatment early: 3 had poor compliance and 2 presented adverse events. Evaluation of treatment efficacy was based on CD4+ T cell enumeration and HIV RNA level quantitation in plasma (NASBA). Baseline values were 278 CD4+/mm3 and 5.42 log RNA copies/ml. Mean changes from baseline were +102 CD4+/mm3 and -2.14 log RNA copies/ml at week 8 and +156 CD4+/mm3 and -2.07 log RNA copies/ml at week 24. HIV RNA in plasma was lower than the detection limit (2.60 log RNA copies/ml) in 55% of patients at week 8 and in 30% at week 24. No major adverse events such as neuropathy or pancreatitis were observed. Once-daily administration of didanosine in combination with twice-daily administration of zidovudine is a well tolerated regimen that appears to be as effective ad the conventional zidovudine/didanosine combination regimen.

Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus.

We report that cells refractory to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV) became susceptible when transfected with a chimeric aminopeptidase-N (APN) cDNA containing a canine domain between residues 643 and 841. This finding shows that APN recognition by these viruses is species related and associated with this C-terminal domain. The human/canine APN chimera was also able to confer susceptibility to the porcine transmissible gastroenteritis virus (TGEV), whereas its human/porcine homolog failed to confer susceptibility to CCV and FIPV. A good correlation was observed between the capacity of CCV, FIPV, and TGEV to recognize the different interspecies APN chimeras and their ability to infect cells derived from the relevant species. As an exception, TGEV was found to use a human/bovine APN chimera as a receptor although itself unable to replicate in bovine cells.

Functional domains in the spike protein of transmissible gastroenteritis virus.

The coronavirus spike protein S is assumed to mediate essential biological functions, including recognition of target cells. Earlier studies from our and other groups identified two regions of the TGEV S (220K) protein possibly implicated in such functions. The first of these corresponds to the 224 amino acid N-terminal region which is deleted in PRCV, the respiratory variant of TGEV. We have examined the pathogenicity for the newborn piglet of a series of neutralization escape mutants encoding an S protein mutated in this region. Several amino acid changes were correlated with a dramatic loss of enterovirulence, thus indicating that crucial determinants are associated with this domain of S. The second region of potential relevance is the major neutralization domain. Baculovirus-vectored expression of 150 to 220 amino acid-long stretches encompassing this region, which is encoded by both TGEV and PRCV, was performed. The resultant recombinant proteins were shown to react with the cognate antibodies and to bind APN specifically, thus localizing the receptor-binding site on the S primary structure. Altogether these data lend support to the view that a domain of S protein structurally distinct from the receptor binding site is required for the virus to express its enteric tropism.