Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe.

Little is known about the genetic relationships between European and other Old-World strains of West Nile virus (WNV) and persistence of WNV North of Mediterranean. We characterized the complete genomes of three WNV strains from France (horse-2000), Tunisia (human-1997) and Kenya (mosquito-1998), and the envelope, NS3 and NS5 genes of the Koutango virus. Phylogenetic analyses including all available full-length sequences showed that: (1) Koutango virus is a distant variant of WNV; (2) the three characterized strains belong to lineage 1, clade 1a; (3) the Tunisian strain roots the lineage of viruses introduced in North America. We established that currently available partial envelope sequences do not generate reliable phylogenies. Accordingly, establishing a large WNV sequence database is pivotal for the understanding of spatial and temporal epidemiology of this virus. For rapid completion of that purpose, colinearized E-NS3-NS5 gene sequences were shown to constitute a valuable surrogate for complete sequences.

[Hantavirus infections in Ile-de-France]. / Infections à Hantavirus en Ile-de-France.

Hantavirus infections, Puumala serotype, is a well-known disease in the northeast of France, but not in the Paris area, despite regularly diagnosed cases. A retrospective study was performed from January 1999 to December 2000 to assess the clinical and epidemiological characteristics of this disease in the "Région Ile-de-France". Fourteen cases were diagnosed. All required hospitalisation. Patients presented usually with high fever, pain, renal failure, moderate thrombocytopenia and, sometimes, transitional acute myopia witch is pathognomonic of the disease. In each case, a contact with a forest was found. Twelve patients recovered completely. One patient with pre-existing chronic renal and hepatic failure died and another developed a persistent arterial hypertension. Invasive procedures were often used before the diagnosis. Hantavirus infections does exist in the "Région Ile-de-France". Failure to recognise this disease in this area lead to unnecessary invasive procedures and hospitalizations.

West Nile outbreak in horses in southern France, 2000: the return after 35 years.

On September 6, 2000, two cases of equine encephalitis caused by West Nile (WN) virus were reported in southern France (Hérault Province), near Camargue National Park, where a WN outbreak occurred in 1962. Through November 30, 76 cases were laboratory confirmed among 131 equines with neurologic disorders. The last confirmed case was on November 3, 2000. All but three cases were located in a region nicknamed "la petite Camargue," which has several large marshes, numerous colonies of migratory and resident birds, and large mosquito populations. No human case has been confirmed among clinically suspected patients, nor have abnormal deaths of birds been reported. A serosurvey has been undertaken in horses in the infected area, and other studies are in progress.

Plasma concentrations of sVCAM-1 and severity of dengue infections.

Adhesion molecules are essential for the immune response. They are involved in the regulation of cell-to-cell contact, thereby enabling leukocytes to communicate. Circulating forms of adhesion molecules are found in the serum of healthy individuals. Raised levels have been associated with disease severity in HCV and other infections and thus appear to be good markers of endothelial damage. The levels of soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) and of sP and sL-selectin in the plasma of children hospitalised for dengue in French Polynesia were monitored. Studies from the 1996/1997 dengue-2 outbreak, showed that levels of sVCAM-1 increase steadily during the febrile period, peak on day 7, and then decline relatively rapidly. Disregarding the time frame within the febrile period, sVCAM-1 levels were always higher compared to controls. There was a significant association between sVCAM-1 levels and dengue haemorrhagic fever, a severe manifestation of dengue virus infection characterised by plasma leakage. No association was apparent between sVCAM-1 levels and primary vs. secondary dengue virus infections. Levels of sP-selectin and sL-selectin were significantly higher in primary compared with secondary infection but were not different in patients presenting with plasma leakage. Lastly, sVCAM-1 levels were significantly higher in an outbreak of severe disease in 1989/1990 (dengue-3) when compared to a non-severe outbreak in 1988/1989 (dengue-1) and a mild outbreak in 1996/1997 (dengue-2). The results suggested that levels of sVCAM-1 production might prove to be a useful marker in the management of severe dengue.

West Nile in the Mediterranean basin: 1950-2000.

Recent West Nile virus (WNV) outbreaks have occurred in the Mediterranean basin. In Algeria in 1994, about 50 human cases of WN encephalitis were suspected, including 8 fatal cases. In Morocco in 1996, 94 equines were affected of which 42 died. In Tunisia in 1997, 173 patients were hospitalized for encephalitis or meningoencephalitis. West Nile serology performed on 129 patients was positive in 111 cases (87%) including 5 fatal cases. In Italy in 1998, 14 horses located in Tuscany were laboratory confirmed for WNV infection; 6 animals died. In Israel in 1998, serum samples from horses suffering from encephalomyelitis had WNV antibodies and virus was isolated from the brain of a stork; in 1999 WNV was identified in commercial geese flocks, and in 2000 hundreds of human cases have been reported. In September 2000, WNV infection was detected in horses located in southern France, close to the Camargue National Park where a WNV outbreak occurred in 1962. By November 30, 76 cases were laboratory confirmed among 131 equines presenting with neurological disorders. No human case has been laboratory confirmed among clinically suspect patients. The virus isolated from a brain biopsy is closely related to the Morocco-1996 and Italy-1998 isolates from horses, to the Senegal-1993 and Kenya-1998 isolates from mosquitoes, and to the human isolate from Volgograd-1999. It is distinguishable from the group including the Israel-1998 and New York-1999 isolates, as well as the Tunisia-1997 human isolate.

Prospective study of the duration and magnitude of viraemia in children hospitalised during the 1996-1997 dengue-2 outbreak in French Polynesia.

The magnitude and duration of viraemia in children admitted to the hospital with dengue was studied during a dengue 2 outbreak in French Polynesia in 1996-1997. Forty-nine patients from whom at least 3 plasma samples were available were included in the study. Based on analysis of IgG-ELISA and haemagglutination inhibition assay, 21 of these were primary and 28 were secondary infections. According to World Health Organization criteria, 42 were dengue fever and 7 were dengue haemorrhagic fever. Virus was detectable by reverse transcription-PCR in all patients for at least the first 3 days of the onset of fever, but was never detected after the 6th day (mean duration = 4.4 days). Plasma virus titers ranged from 1.7-5.6 Log(10) TCID(50)/ml. A significant difference was not observed in the magnitude and duration of viraemia in patients with primary versus secondary infections. The severity of the illness, however, was correlated with both criteria.

Dengue: an evaluation of dengue severity in French Polynesia based on an analysis of 403 laboratory-confirmed cases.

We conducted a retrospective study of 403 laboratory-confirmed dengue cases hospitalized in Tahiti between August 1989 and March 1997. According to standard WHO criteria, 337 of these cases were dengue fever (DF) and 64 were dengue haemorrhagic fever (DHF). Of the 10 fatal cases, 6 were DF and 4 were DHF. As an alternative, we used a correspondence analysis procedure to define dengue severity based on basic clinical and biological criteria for which we assigned a severity score, and then selected the 50 most severe cases from this analysis. Of the latter, 17 patients had been classified as DF and 33 as DHF by the WHO criteria. From this analysis, haemorrhages and decreased platelets counts associated with hepatic disorders are the main criteria associated with the severe dengue cases. Thus in our study population, the WHO classification does not account for the overall severity of dengue; hepatic failure should be considered as a specific severe form of dengue since plasma leakage, which is the pathophysiological hallmark of DHF, is only one of the pathogenic mechanisms leading to severity.

Changing clinical and biological manifestations of dengue during the dengue-2 epidemic in French Polynesia in 1996/97--description and analysis in a prospective study.

In August 1996 dengue-2 virus was detected in French Polynesia for the first time since 1976. A prospective study was conducted from November 1996 to April 1997. Each time one of 7 physicians suspected dengue, the patient was enrolled and epidemiological, clinical and biological data were recorded. Dengue diagnosis was confirmed by virus isolation and IgM detection. The aims of this study were to find clinical and biological predictive factors constituting a specific profile of dengue (DF) and dengue haemorrhagic fever (DHF/DSS) and to assess the possibility of diagnosing dengue at primary health care level using clinical criteria and basic laboratory parameters. Of 298 clinically suspect cases, 196 (66%) were confirmed as dengue. The association of macular rash, pruritus, low platelet count and leukopenia was statistically predictive of dengue but not clinically, since these four signs occur in many other viral infections. As the prevalence of clinical and biological manifestations varied over time in our study, a specific profile useful for dengue diagnosis cannot be defined. With six cases of DHF, the morbidity of this dengue-2 outbreak was very low despite the sequential infection scheme DEN-3/DEN-2. The clinical expression of dengue could depend on a specific virus strain circulating in a specific population in a particular place, with varying virulence over time.

Possible dengue sequential infection: dengue spread in a neighbourhood during the 1996/97 dengue-2 epidemic in French Polynesia.

A DEN-2 epidemic occurred in French Polynesia from August 1996 to April 1997 after 7 years of DEN-3 circulation. The susceptible population constituted all expatriates and Polynesians under 21. In August 1996, two successive DEN-2 cases occurred in Teroma, a Tahitian neighbourhood close to the international airport of Tahiti. A serological prospective study of persons < 21 years living in Teroma was conducted. The study population was bled in September 1996, October 1996 and June 1997. Analysis of dengue spread in Teroma confirmed that dengue transmission occurs primarily in the house, thus vector control campaigns should incorporate focal insecticide spraying and systematic daily use of insecticide in houses. The evolution in time of the disease demonstrated that among a susceptible population, prevalence and incidence rates are related to the time of exposure, and consequently to age. Comparison of dengue incidence or dengue prevalence between populations therefore requires adjusted age rates. Most studies did not adjust for age, leading to the conclusion that DHF is more frequent during secondary than during primary dengue infection. Prospective studies taking into account the time of dengue exposure are necessary to confirm the sequential infection hypothesis.

[The specific epidemiological surveillance of dengue: the method and its importance since the dengue-2 epidemic in French Polynesia in 1996]. / Surveillance épidémiologiqe specifique de la dengue: méthode et intérêt lors de l'épidémie de dengue 2 en Polynésie française en 1996.

Dengue fever is present in tropical and subtropical regions and its geographical extension and the simultaneous increase of its mortality are worrisome. In endemic or epidemic countries, the aim of dengue-specific epidemiological surveillance is to confirm as soon as possible the circulation of a new viral dengue serotype, i.e. the beginning of an epidemic. The efficiency of the control strategy is improved by an earlier epidemic alert. In French Polynesia, dengue-3 virus circulated since 1989 at low level and, in May 1996, a specific epidemiological surveillance was undertaken because of the threat of a dengue-4 epidemic. From each suspected dengue case reported by 18 Polynesian physicians located in the Société Islands, a blood sample was taken for virological assay and clinical data were reported. Between May and November 1996, the virology unit of the Institut Malardé isolated 21 viruses (2 dengue-3 and 19 dengue-2) from 302 suspected cases. The dengue-specific epidemiological surveillance confirmed that dengue-2 virus was circulating and reduced the time of the epidemiological alert by 2 or 3 months compared to previous epidemics. Taking into account the day of illness, a logistic regression undertaken on the clinical data showed that the absence of cough was the only predictive sign of dengue diagnosis. The performance of this dengue-specific epidemiological surveillance system led us to consider its implementation in all concerned countries. A collaboration with international reference laboratories could be a solution for the developing countries.

Implication of macrophage inflammatory protein-1alpha in the inhibition of human haematopoietic progenitor growth by dengue virus.

The mechanisms were investigated of haematopoietic progenitor growth inhibition, observed after in vitro infection of cord blood mononuclear cells (CBMNC) by a clinical isolate of dengue 3 (29-56DSS). The level of virus replication was not different when CBMNC were inoculated with 29-56DSS compared with a prototype strain of dengue 3 (H-87) which had no inhibitory effect. An inhibitory effect was also observed when cell-free and heat-inactivated supernatants from 29-56DSS cultures, but not from H-87 cultures, were added to cultures of normal CBMNC, suggesting an indirect mechanism via the release of soluble suppressive factor(s). Macrophage inflammatory protein-1alpha (MIP-1alpha) was detected at a significantly higher level in 29-56DSS cultures than in controls. Blocking experiments with anti-MIP-1alpha antibody demonstrated that the inhibitory effect was related at least partly to high MIP-1alpha levels. To our knowledge, this is the first report suggesting an indirect effect of dengue infection on haematopoiesis mediated by a suppressive cytokine.

Plasma levels of tumour necrosis factor alpha and transforming growth factor beta-1 in children with dengue 2 virus infection in French Polynesia.

The pathogenesis of dengue haemorrhagic fever (DHF) is not well understood. In the absence of predictive clinical or biological criteria, the management of DHF patients remains difficult. The role played by cytokines in the occurrence of DHF has been suggested by several authors. In this study, we determined the plasma levels of tumour necrosis factor alpha (TNF alpha) and transforming growth factor beta-1 (TGF beta-1) in 52 children with laboratory-confirmed dengue virus infection admitted to hospital during the recent dengue 2 outbreak in French Polynesia. Thirty-three children were classified as having dengue fever (DF) and 19 as DHF. The plasma of both DF and DHF patients contained similar levels of TNF alpha. By contrast, plasma obtained from children with DHF had significantly higher levels of TGF beta-1 than plasma from children with DF, especially from days 1 to 3 after the onset of fever.

Dengue virus inhibits human hematopoietic progenitor growth in vitro.

Dengue disease, whether it be classical dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS), is frequently associated with hematologic disorders. The underlying cause of these abnormalities is unknown. To determine if an inhibitory effect on human hematopoietic progenitor growth can be observed, normal cord blood mononuclear cells were exposed to low-passaged clinical isolates from DF, DHF, and DSS patients and to the prototype strain of dengue-3 virus (H-87). In primary methylcellulose cultures, there was no inhibition of colony formation. After an initial 8-day liquid culture, inhibition was observed with the isolates, but strain H-87 had no effect. Furthermore, isolates from patients with DSS showed a more potent inhibitory effect. These data represent the first documented study of in vitro impaired progenitor cell growth by dengue virus and suggest that this inhibition could be dependent upon the isolate tested.

Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium.

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.

Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.

Trefoil peptides are a recently recognized group of small peptides abundantly produced at mucosal surfaces that offer the opportunity to define mechanisms of mucosal cell-specific differentiation and to illuminate new mechanisms for the preservation of mucosal integrity. We report the cDNA cloning of a 75-amino acid human trefoil factor expressed in small and large intestinal mucosas that is highly homologous to the intestinal trefoil factor, with 70% identity at the amino acid level of the predicted mature protein. This human intestinal trefoil factor is also homologous, although to a lesser extent, to trefoil peptides expressed at other sites in the gastrointestinal tract in man, exhibiting absolute conservation of the P domain motif (CX9CX9CX4CCX9WCF) that defines this family of peptides. These findings indicate a high degree of evolutionary conservation of organ/region-specific members of this peptide family. In situ hybridization of intestinal trefoil factor demonstrates a high degree of expression in mature small intestine villus and colonic epithelial goblet cells. Immunogold staining demonstrates high concentrations of intestinal trefoil factor in the rough endoplasmic reticulum and theca of goblet cells as well as throughout the mucosal surface, consistent with vectorial secretion of this factor by goblet cells onto the intestinal luminal surface. In addition, intestinal trefoil factor was also localized within columnar epithelial cells by immunogold labeling despite the absence of mRNA. These observations suggest that peptide secreted by goblet cells might be taken up from the luminal surface and transcytosed by enterocytes. Human intestinal trefoil factor expression was also detected in the HT-29N2 and HT-29H2 subclones in conjunction with the emergence of the goblet cell phenotype, but not in the CaCO2 cell line that exhibits enterocytic phenotype. In summary, these findings confirm the existence of a highly conserved family of peptides that are abundantly expressed in distinctive regions throughout the gastrointestinal tract in a highly cell-specific pattern reflecting a goblet cell differentiation pathway. They form one of the more abundant constituents of the interface between the mucosa and "outside" environment and may provide a new paradigm of regulation of the integrity of epithelial surfaces as well as a previously unrecognized dimension of goblet cell function.

Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction.

alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.

Fatty acylation of human platelet proteins: evidence for myristoylation of a 50 kDa peptide.

Fatty acid acylation of platelet proteins was studied by measuring incorporation of [3H]palmitate and [3H]myristate after incubation at 37 degrees C for 4 h. About ten major radiolabeled proteins were detected after SDS-polyacrylamide gel electrophoresis and fluorography, for both fatty acids. Cleavage by hydroxylamine treatment indicated an ester bond of either palmitate or myristate to these proteins. Nevertheless, a single 50 kDa peptide was specifically modified by an amide-linked myristate. The functions of acylated proteins in platelets are still unknown, but their relation with DLPC-induced shape changes and vesicle shedding is excluded.