Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Identification of helper T cell epitopes of dengue virus E-protein.

The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides. Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice. Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E. coli as polypeptides fused to trpE (f22-205, f267-354, f366-424). To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes. Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359. Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes. Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice. This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design.

Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection.

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.

Identification of Ebola virus sequences present as RNA or DNA in organs of terrestrial small mammals of the Central African Republic.

The life cycle of the Ebola (EBO) virus remains enigmatic. We tested for EBO virus in the organs of 242 small mammals captured during ecological studies in the Central African Republic. EBO virus glycoprotein or polymerase gene sequences were detected by reverse transcription PCR in RNA extracts of the organs of seven animals and by PCR in DNA extract of one animal. Neither live virus nor virus antigen was detected in any organ sample. Direct sequencing of amplicons identified the virus as being of the Zaire/Gabon subtype. Virus-like nucleocapsids were observed by electron microscopy in the cytoplasm of the spleen cells of one animal. The animals belonged to two genera of rodents (Muridae; Mus setulosus, Praomys sp1 and P. sp2) and one species of shrew (Soricidae; Sylvisorex ollula). These preliminary results provide evidence that common terrestrial small mammals living in peripheral forest areas have been in contact with the EBO virus and demonstrate the persistence of EBO virus RNA and DNA in the organs of the animals. Our findings should lead to better targeting of research into the life cycle of the EBO virus.

Enhanced TNF alpha production by monocytic-like cells exposed to dengue virus antigens.

The studies indicating the importance of TNF alpha in dengue virus infection have led us to determine whether monocyte-like cells produce TNF alpha exposure after dengue virus. The supernatant fluids of mosquito cells (AP61) infected with dengue virus (DV) type 1 and DV type 3 were harvested 7 days post-infection and clarified. DV inactivation was performed in the presence of betapropiolactone that preserves antigenicity of viruses. We used the monocytic-like cell line THP-1 that is a model system of TNF alpha production. Polymyxin B (50 micrograms/ml) was added to block untoward effects resulting from possible LPS contamination of media or cultures. THP-1 cells were primed with a phorbol ester (PMA) for 24 h, then they were cultured for 4 and 24 h in the presence of inactivated culture supernatant of dengue infected AP61 cells or control preparations. The concentrations of TNF alpha in the culture supernatants were measured by using an immunoenzymatic assay. PMA-treated THP-1 cells rapidly secreted TNF alpha in response to inactivated culture supernatant of DV-infected cells. We found high levels of TNF alpha with cells exposed to DV1 and DV3 preparations compared with controls (mean values; 465 and 829 vs. 70 pg/ml, respectively, at 24 h post exposure, n = 4). We obtained a substantial inhibition of the enhancing activity of DV1 and DV3 infected supernatants in the presence of dengue hyperimmune mouse ascitic fluids. Our results demonstrate that exposure of monocytes/macrophages to DV particles or virus proteins derived from DV may be responsible for the enhanced production of TNF alpha in DV-infected patients.

Report of a fatal case of dengue infection with hepatitis: demonstration of dengue antigens in hepatocytes and liver apoptosis.

A fatal case of dengue (DEN) infection associated with a spleen rupture and with hepatitis is reported here. Microscopic studies showed numerous areas of spleen rupture with hematomas and revealed necrotic foci in liver samples obtained at autopsy. Although hepatitis was reported in several cases of DEN fever, the mechanism of liver injury remains poorly understood. In this case, immunohistochemistry showed that DEN viral antigens were mostly detected in hepatocytes surrounding the necrotic foci. By in situ detection of DNA fragmentation, apoptotic hepatocytes were found to be colocated with DEN virus-infected hepatocytes. These findings suggest that hepatocytes are the major sites of DEN virus replication in the liver and that DEN virus induces apoptosis of hepatocytes in vivo.

Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children.

We studied five fatal cases of dengue haemorrhagic fever (DHF), confirmed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method, in Vietnamese children. The liver seems to be a target for dengue virus, so postmortem examinations were performed to investigate elementary lesions, local recruitment of inflammatory cells and whether the virus was present in target cells of the liver. We detected severe, diffuse hepatitis with midzonal necrosis and steatosis in two patients, focal areas of necrosis in two patients, and normal histology in one patient. Dengue virus antigen was detected using immunohistochemistry in hepatocytes from necrotic areas in four cases. There was no recruitment of polymorphonuclear cells, and no lymphocytes were detected in the liver lesions of patients who died from DHF. Lymphocytic infiltration occurred in only one hepatitis B virus-positive patient, with no signs of chronic hepatitis. Kupffer cells had mostly been destroyed in cases with focal or severe necrosis. TUNEL tests were positive in necrotic areas, with positive cells forming clusters, suggesting that an apoptotic mechanism was involved. Thus, we suggest that the hepatocyte and Kupffer cells may be target cells supporting virus replication and that the councilman body is an apoptotic cell, as in the pathogenesis of yellow fever.

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.

Variations in biological features of West Nile viruses.

Pathological findings in humans, horses, and birds with West Nile (WN) encephalitis show neuronal degeneration and necrosis in the central nervous system (CNS), with diffuse inflammation. The mechanisms of WN viral penetration of the CNS and pathophysiology of the encephalitis remain largely unknown. Since 1996, several epizootics involving hundreds of humans, horses, and thousands of wild and domestic bird cases of encephalitis and mortality have been reported in Europe, North Africa, the Middle East, Russia, and the USA (see specific chapters in this issue). However, biological and molecular markers of virus virulence should be characterized to assess whether novel strains with increased virulence are responsible for this recent proliferation of outbreaks.

Morphogenesis of yellow fever virus in Aedes aegypti cultured cells. I. Isolation of different cellular clones and the study of their susceptibility to infection with the virus.

We report the isolation of 19 cellular clones of Aedes aegypti and analyze their susceptibility to infection with yellow fever virus, in comparison with the uncloned cells. Four types of clones were found, different in their morphology, their metabolism and their virus production. The C 17 clone produces the most virus, not only with respect to the other Aedes aegypti clones, but also to other uncloned arthropod cells published in the literature. A cytopathic effect exists in the virus infected cells. Cloned and uncloned cells do not seem to produce any antiviral substance which transfers to other cells an immunity against the virus. An immunological study with immunoperoxidase enabled us to follow the appearance of viral antigens and to locate them in the cell. At the beginning we observed a perinuclear coloration diffusing further into the whole cytoplasm. The present report represents an introduction to a structural study on the morphogenesis of yellow fever virus within the cells of Aedes aegypti.

Genetic study of Japanese encephalitis viruses from Vietnam.

Two hundred eighty-five nucleotides from the capsid/premembrane gene region of 16 isolates of Japanese encephalitis (JE) virus from the southern and northern regions of Vietnam obtained during the period 1964-1988 were sequenced and compared with each other and with wild JE virus strains from China and Japan. The 16 Vietnamese isolates showed low rates of genetic evolution (< or = 3.2%) and were classified into one genotype with an overall similarity of > or = 95.4%, including the Japanese JaArOS982 strain taken as reference, and earlier isolated strains from Japan (Nakayama 1935) and China (Beijing-1 1949 and SA14 1954), the wild-type strains used to derive JE vaccines. The implications of the close genetic relationship among Vietnamese strains of JE virus analyzed in our study in relation to their epidemiology and evolution are discussed.

Detection of dengue virus RNA by reverse transcription-polymerase chain reaction in the liver and lymphoid organs but not in the brain in fatal human infection.

Autopsy tissues from 18 children believed to have died of dengue hemorrhagic fever were tested for the presence of dengue virus RNA by reverse transcription-polymerase chain reaction (RT-PCR). Such RNA was found in 14 of 18 liver specimens, 13 of 18 spleen specimens and 7 of 16 mesenteric lymph node specimens. No dengue virus RNA was detected in 44 samples of brain tissue from 15 individuals, 1 or more of whose other tissues yielded such RNA. All tissues had been tested previously for dengue virus by mosquito inoculation. In those tests, virus was recovered from 5 of 18 liver and 2 of 18 spleen specimens. Thus, the RT-PCR is more sensitive than the most sensitive virus isolation technique for detecting dengue virus or its components in human tissue. Failure to isolate virus from most of spleen and all mesenteric lymph node specimens may indicate that those tissues contained primarily degraded virus undergoing inactivation.

Morphogenesis of yellow fever virus in Aedes aegypti cultured cells. II. An ultrastructural study.

The growth and intracytoplasmic development of two yellow fever virus strains (wild and French neurotropic) were studied in Aedes aegypti cells (clone C 17). Despite a longer period of latency for the vaccine virus, infected cells appeared similar. The cisternae of the rough endoplasmic reticulum (RER) were swollen and formed vesicles which contained the virus. This RER appeared to be the predominant locus of viral synthesis and maturation. Cytopathic effect appeared when the cells were filled with vesicles, and it was characterized by cell degeneration and lysis.

Partial nucleotide and amino acid sequences of the envelope and the envelope/nonstructural protein-1 gene junction of four dengue-2 virus strains isolated during the 1981 Cuban epidemic.

In 1981, an epidemic of dengue hemorrhagic fever (DHF) caused by dengue-2 virus occurred in Cuba. This was the first DHF epidemic reported in the Western Hemisphere. In this study, we have analyzed four dengue-2 Cuban strains for two short genomic fragments: one on the envelope (E) glycoprotein and one at the E/nonstructural protein-1 (NS1) gene junction. The E segment of these 1981 Cuban isolates were more closely related to older dengue-2 virus strains such as New Guinea C 1944, Thailand 1964, Sri Lanka 1968, and Burma 1976 than to more recent isolates of this virus from Jamaica and Vietnam. More than 9% of the divergence with strains isolated from Jamaica and Vietnam was observed at the E/NS1 gene junction. One nucleotide change was observed between the first strain isolated during the epidemic and the rest of the Cuban strains. This mutation induced a nonconserved amino acid change from phenylalanine to leucine at position 43 that was not observed in any of the other strains with which it was compared.

The first epidemic of dengue hemorrhagic fever in French Guiana.

From July 1991 to October 1992, an outbreak of dengue spread into the main urban areas of French Guiana, where 90% of the country's 114,808 inhabitants live. In mid-July 1991 dengue-2 virus was identified as being responsible for most cases, while dengue-1 virus was rarely isolated and circulated at a low level. The number of dengue cases during this period was unknown because there was no clinically based dengue surveillance system. The only available data were for the number of suspected cases as indicated by the number of patients for whom blood samples were submitted to a laboratory for dengue diagnosis. Eight hundred forty-seven of the 2,948 suspected cases were diagnosed in the laboratory as dengue cases. Six fatal cases were reported. This outbreak was marked by the appearance of the first clinical cases of dengue hemorrhagic fever (DHF) in French Guiana. Forty cases met the World Health Organization definition of clinical DHF: 32 were grade II, seven were grade III, and one was grade IV and fatal. Eighteen cases were confirmed in the laboratory and 12 were probable; there was no proof of the dengue etiology for the remaining patients.

Comparison of the enzyme-linked immunosorbent assay (ELISA) with standard tests used to detect yellow fever virus antibodies.

The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to yellow fever virus in 110 sera from patients living in an epidemic yellow fever area. The results were then compared with those obtained with the hemagglutination-inhibition (HI), complement-fixation (CF), neutralization (NT), and indirect immunofluorescence (IFA) tests. This ELISA, which used a type-specific antigen, showed the same results as the NT test and was found to be more sensitive and more specific than the HI and CF tests.

Homogeneity among Senegalese strains of yellow fever virus.

A series of 16 yellow fever (YF) viruses isolated from mosquitoes, monkeys and humans in different epidemiological contexts in Senegal and The Gambia between 1976 and 1983, was analyzed by T1 RNase oligonucleotide fingerprints of the genomic 32P-labeled RNA, by SDS-polyacrylamide gel electrophoresis of the intracellular virus-specified polypeptides, by peptide mapping of the envelope E glycoprotein and by immunological reactivities with monoclonal antibody fluids (MAF's) against the E glycoprotein. These strains had not been passed in suckling mice and were isolated in Aedes pseudoscutellaris Mos 61 cultured cells. These strains showed no virulence in three-week-old Swiss mice when injected intraperitoneally. Direct comparison of the large T1 RNase-resistant oligonucleotide maps indicated a relative genetic stability (92%-100%). A greater change was observed when these strains were compared with an epidemic YF strain isolated in 1965 with an oligonucleotide fingerprint map sharing 82%-88% similarity. The YF-specified proteins were identical in their molecular weight, and the fragments obtained after limited proteolysis of the envelope protein using protease V8 or alphachymotrypsine indicated that the strains were chemically similar. Only a few differences were observed when the strains were seroneutralized with MAF's, but no relation could be made with genetic or biological data. This suggested that the YF virus strains isolated from the same geographic area and during a short period of time had evolved slowly. Moreover, all the viruses were closely related and no correlation could be established with the apparent variations in virulence in nature.

Dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein.

The maltose-binding protein (MalE) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. In this study, MalE was produced in Spodoptera frugiperda (Sf9) lepidopterian cells using the baculovirus expression system. The secretion of MalE, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected Sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 micrograms per 10(6) cells) of highly purified protein. The gene encoding the envelope glycoprotein E of the dengue (DEN) type 2 virus deleted of its C-terminal 102 amino acids (D2E delta 102) was fused to the MalE gene. The resulting hybrid MalE-D2E delta 102 glycoprotein was processed through the Golgi network of Sf9 cells and was secreted. It was retained on a maltodextrin column and was eluted with maltose. Antigenic and immunogenic properties dependent on the three-dimensional structure in the native E protein were preserved in the recombinant MalE-D2E delta 102 protein. Thus MalE with its signal sequence may be used as a carrier protein for production in the baculovirus system and purification of proteins which require transportation through intracellular compartments for correct folding and processing.

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Tick-borne virus diseases of human interest in Europe.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.