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purified ns2b/ns3 serine protease of dengue virus type 2 exhibits cofactor ns2b dependence for cleavage of substrates with dibasic amino acids in vitro.dengue virus type 2 ns3, a multifunctional protein, has a serine protease domain (ns3pro) that requires the conserved hydrophilic domain of ns2b for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. in this study, we report the expression of the ns2b-ns3pro precursor in escherichia coli as a fusion protein with a histidine tag at the n terminus. the precursor was purified from insoluble inclusion bodies by ni(2+) affinity and gel filtration chro ...200010744671
chimeric dengue type 2 (vaccine strain pdk-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.we constructed chimeric dengue type 2/type 1 (den-2/den-1) viruses containing the nonstructural genes of den-2 16681 virus or its vaccine derivative, strain pdk-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of den-1 16007 virus or its vaccine derivative, strain pdk-13. we previously reported that attenuation markers of den-2 pdk-53 virus were encoded by genetic loci located outside the structural gene region of the pdk-53 virus genome. chi ...200010708416
cis- and trans-acting elements in flavivirus rna replication.most of the seven flavivirus nonstructural proteins (ns1 to ns5) encoded in the distal two-thirds of the rna positive-sense genome are believed to be essential components of rna replication complexes. to explore the functional relationships of these components in rna replication, we used trans-complementation analysis of full-length infectious rnas of kunjin (kun) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repbhk cell line stably produci ...200010708442
gb virus c/hepatitis g virus infection is frequent in american children and young adults.the novel flavivirus gb virus c/hepatitis g virus (gbv-c/hgv) has been detected in approximately 2% of blood donors in the united states, and neutralizing antibody to the envelope protein (e2), a marker of previous infection with gbv-c/hgv, is present in approximately 9% of donors. the rate of gbv-c/hgv infection among american children is unknown. to determine whether viral infection might occur during childhood, 160 serum specimens (obtained from blood bank samples) from children and young adu ...200010722444
japanese encephalitis dna vaccine candidates expressing premembrane and envelope genes induce virus-specific memory b cells and long-lasting antibodies in swine.swine are an important amplifier of japanese encephalitis (je) virus in the paradomestic environment. in this study, two je dna vaccine candidates were evaluated for immunogenicity in swine. both vaccine plasmids encode a cassette consisting of the signal of premembrane (prm), prm, and envelope (e) coding regions of je virus. one plasmid, designated pcjeme, is based on a commercial vector (pcdna3), whereas the other plasmid, designated pnjeme, is based on a vector (pngvl4a) designed to address s ...200010683326
immunisation with gamma globulin to murray valley encephalitis virus and with an inactivated japanese encephalitis virus vaccine as prophylaxis against australian encephalitis: evaluation in a mouse model.in northwestern australia, the flavivirus murray valley encephalitis (mve) poses a significant health risk to infants in some aboriginal communities, particularly during each wet season. while there are too few cases to warrant the development of a vaccine against mve, a safe, effective prophylaxis for these children is still urgently required. the use of passive transfer of human gamma globulin to mve or immunisation with a vaccine to the closely related japanese encephalitis (je) virus were in ...200010797383
[west nile fever].reviews the data on the history, distribution, ecology, epidemiology, and clinical picture of west nile (wn) fever and the ecology of the virus (flaviviridae, flavivirus, japanese encephalitis complex). analyzes the causes of sudden epidemic outbreaks with high mortality in rumania in 1996, in european southern russia, and in new york and its environs in 1999. offers recommendations on decreasing the damage inflicted by new and emerging infections leading to epidemic emergencies.200010765542
[the immune state against yellow fever vaccinal virus (17d) in 2 populations of bahia state, brazil].the occurrence of cases of yellow fever (yf) and also the extensive distribution of a. aegypti in brazil, inspired a study about the estimate of immunity against vaccinal virus (17d) among the residents at two cities of the bahia state, ipupiara (n = 461) and prado (n = 228). at this non-endemic area of yf, the search for serology antibody against 17d (ab17d) and 18 another arbovirus was made thereby hemagglutination inhibition (hi). only 1.2% (8/689) showed ab17d, six of those with monotypic so ...200010881117
human skin langerhans cells are targets of dengue virus infection.dengue virus (dv), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. macrophages are important in dengue pathogenesis; however, the initial target cell for dv infection remains unknown. as dv is introduced into human skin by mosquitoes of the genus aedes, we undertook experiments to determine whether human dendritic cells (dcs) were permissive for the growth of dv. initial experiments demonstrated that blood-derived dcs were 10-fold ...200010888933
definition of an epitope on japanese encephalitis virus (jev) envelope protein recognized by jev-specific murine cd8+ cytotoxic t lymphocytes.we defined an epitope on the japanese encephalitis virus (jev) envelope (e) protein recognized by cd8+ cytotoxic t lymphocytes (ctls). ctls induced in jev-infected balb/c (h-2d) mice recognized e and/or premembrane (prm) proteins, while ctls in c57bl/6j (h-2b) and c3h/hej (h-2k) mice did not. jev-specific ctls had a phenotype of cd3+ cd4- cd8+. twenty-four 9-amino acid (a.a.) peptides, which had binding motifs for h-2kd, h-2ld or h-2dd, were synthesized according to the amino acid sequences of p ...200010795519
processing of gb virus b non-structural proteins in cultured cells requires both ns3 protease and ns4a cofactor.the identification of antivirals and vaccines against hepatitis c virus (hcv) infection is hampered by the lack of convenient animal models. the need to develop surrogate models has recently drawn attention to gb virus b (gbv-b), which produces hepatitis in small primates. in a previous study in vitro, it was shown that gbv-b ns3 protease shares substrate specificity with the hcv enzyme, known to be crucial for virus replication. in this report, gbv-b ns3 activity on gbv-b precursor proteins has ...200010950975
[tick-borne meningoencephalitis in thurgau canton: a clinical and epidiomological analysis].tick-borne meningo-encephalitis (tbe) is caused by a flavivirus species that is transmitted from rodents to humans by ixodes ticks. the clinical picture of central nervous system involvement varies from febrile headache to lethal encephalitis. to date no specific treatment exists, although protective active immunisation is available.200011075408
trends in flavivirus infections in japan.although japanese encephalitis has declined as an important cause of illness and death in japan, infection with other flaviviruses has become a public health concern. recently, reports of imported dengue cases, as well as isolations of tick-borne encephalitis virus, have increased.200011076713
dengue infection presenting with central nervous system manifestation.the objective of this study was to investigate the possibility of dengue virus infection causing an abnormal neurologic presentation. between 1996 and 1998, all pediatric patients with clinical manifestations of encephalitis-like illness who were admitted to the department of pediatrics, siriraj hospital were prospectively studied for any evidence of dengue virus infection. the diagnosis of dengue virus infection was based on mosquito viral isolation and serologic and polymerase chain reaction ( ...200010961794
crystal structure of dengue virus ns3 protease in complex with a bowman-birk inhibitor: implications for flaviviral polyprotein processing and drug design.dengue viruses are members of the flaviviridae and cause dengue fever and the more severe dengue hemorrhagic fever. although nearly 40 % of the world's population is at risk of dengue infection, there is currently no effective vaccine or chemotherapy for the disease. processing of the dengue polyprotein into structural and non-structural proteins in a host, which is essential for assembly of infective virions, is carried out by the combined action of host proteases and the trypsin-like, two-comp ...200010966782
modulatory effects of the human heat shock protein 70 on dna vaccination.dna vaccination with the plasmid expressing japanese encephalitis virus (jev) nonstructural protein 1 (pjns1) has been shown to induce effective immunity against jev infection. to further increase the efficacy of pjns1 dna vaccination, we coinjected pjns1 with a plasmid that expresses heat shock protein 70.1 (phsp70.1) into mice. we found that coinjection of phsp70.1 enhanced both t cell proliferation and cytotoxic effects, but not the antibody response to jev. moreover, mice immunized with both ...200010971139
substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.the flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (fg loop) in the envelope (e) protein. in some flaviviruses this domain harbors the integrin-binding motif arg-gly-asp (rgd). one of us has shown earlier that host cell adaptation of murray valley encephalitis virus (mve) can result in the selection of attenuated variants altered at e protein residue asp(390), which is part of an rgd motif. here, a full-length, infectious cdna clone of mve was constructed ...200010982329
full-length gb virus c (hepatitis g virus) rna transcripts are infectious in primary cd4-positive t cells.gb virus c (gbv-c or hepatitis g virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. although gbv-c is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. we constructed a full-length cdna from the plasma of a person with chronic gbv-c viremia. peripheral blood mononuclear cells (pbmcs) transfected with full-length rna transcripts from this gbv-c clone resulted in viral replication. this was demonstrat ...200010982359
gb virus c/hepatitis g virus replicates in human haematopoietic cells and vascular endothelial cells.a novel flavivirus, gb virus c (gbv-c)/hepatitis g virus (hgv), has been detected in chronic liver disease patients. it is known that the viral rna can be detected in approximately 5% of american blood donors. however, the implications for liver disease and the sites of virus replication remain unknown. possible sites of virus replication were studied by using cell lines and/or primary cells derived from human lymphoid cells, myeloid cells, hepatocytes and endothelial cells. rna was detected by ...200010993934
yellow fever virus ns2b-ns3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function.charged-to-alanine substitutions and deletions within the yellow fever virus ns2b-ns3(181) protease were analyzed for effects on protease function. during cell-free translation of ns2b-3(181) polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single n-terminal cluster in the conserved domain of ns2b (residues elkk(52-55)) and two in ns3 (ed(21-22), and residue h(47)). these mutations inhibited other protease-dependent cleavages of a transiently expressed ...200010998334
solid phase synthesis of aminoboronic acids: potent inhibitors of the hepatitis c virus ns3 proteinase.use of a resin bound diol as a boronic acid protecting group has been developed to allow the parallel synthesis of potent inhibitors of the hepatitis c virus ns3 serine proteinase.200010915055
attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site.the impact of a specific region of the envelope protein e of tick-borne encephalitis (tbe) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. the four amino acid residues that were analyzed in detail (e308 to e311) are located on the upper-lateral surface of domain iii according to the x-ray structure of the tbe virus protein e and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. mutants containing si ...200011000232
a model for the hepatitis c virus envelope glycoprotein e2.several experimental studies on hepatitis c virus (hcv) have suggested the envelope glycoprotein e2 as a key antigen for an effective vaccine against the virus. knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. this paper reports the application of fold recognition methods in order to produce a model of the hcv e2 protein. such investigation highlighted the envelope protein e of tick borne encephalitis virus as a possible template ...200010861927
role of the dexh motif of the japanese encephalitis virus and hepatitis c virus ns3 proteins in the atpase and rna helicase activities.the role of the conserved dexh motif of the japanese encephalitis virus (jev) ns3 protein in the atpase and rna helicase activities was compared with that of the hepatitis c virus (hcv) ns3 protein. in the dexh motif of jev ns3, asp-285 and glu-286 were essential for both atpase and rna helicase activities. cys-287 was critical for the rna helicase activity of jev ns3 but not for atpase activity. a his-288-to-ala substitution in the dexh motif of hcv ns3 resulted in an increase in atpase activit ...200010915602
gbv-c/hgv genotypes: proposed nomenclature for genotypes 1-5.the gb virus-c and hepatitis g virus (gbv-c/hgv) are variants of the same flavivirus. this proposal attempts to clarify the conflicting nomenclature for gbv-c/hgv genotypes. the first three genotypes described were genotype 1 (west africa); genotype 2 (us/europe) and genotype 3 (asia). subsequently, two groups published data from south africa and southeast asia both stating the presence of a novel "4th genotype." these isolates are distinct phylogenetically. it is proposed that the nomenclature ...200010935992
ns3 serine protease of bovine viral diarrhea virus: characterization of active site residues, ns4a cofactor domain, and protease-cofactor interactions.the gene expression of bovine viral diarrhea virus (bvdv), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. a protease located in the n-terminal region of nonstructural (ns) protein ns3 catalyzes the cleavages, leading to the release of ns4a, ns4b, ns5a, and ns5b. our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute togethe ...200010915606
prospective follow-up of patients with gbv-c/hgv infection: specific mutational patterns, clinical outcome, and genetic diversity.an association between a specific mutational pattern within the nonstructural (ns)3 region of gb virus-c/hepatitis g virus (gbv-c/hgv) genome and fulminant hepatic failure has been suggested recently. the mutational pattern consists of 3-6 nucleotide mutations of which one is leading to an amino acid exchange. in the present study, patients with gbv-c/hgv mono-infection (n = 24) or gbv-c/hgv and hcv co-infection (n = 20) were investigated prospectively. in 6/44 patients (14%) the mutational patt ...200011002248
single amino acid differences are sufficient for cd4(+) t-cell recognition of a heterologous virus by cattle persistently infected with bovine viral diarrhea virus.cattle that are persistently infected (pi) with one strain of bovine viral diarrhea virus (bvdv) can resolve infection with a second, antigenically heterologous strain but not the homologous strain. since cd4(+) t cells are thought to be critical for the resolution of acute bvdv infection (howard et al., 1992, vet. immunol. immunopathol. 32, 303-314), we have examined the recognition of a heterologous virus (nadl) by cd4(+) t cells from pe515-pi animals. the immune response of non-pi control cat ...200011021996
[circulation of virus and interspecies contamination in wild animals].paradoxically, just when we have succeeded in eradicating and/or bringing under control the major viral infections (smallpox, poliomyelitis, measles) numerous viral infections are emerging in man and in animals. changes in our social environment, technological and ecological equilibrium have facilitated this phenomenon. furthermore, certain of these viruses have demonstrated an almost unlimited capacity to adapt genetically to environmental change. hiv has already infected 40 million individuals ...200011030047
chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific cd8 t-cell response.we have constructed a chimeric yellow fever/dengue (yf/den) virus, which expresses the premembrane (prm) and envelope (e) genes from den type 2 (den-2) virus in a yf virus (yfv-17d) genetic background. immunization of balb/c mice with this chimeric virus induced a cd8 t-cell response specific for the den-2 virus prm and e proteins. this response protected yf/den virus-immunized mice against lethal dengue encephalitis. control mice immunized with the parental yfv-17d were not protected against de ...200010933719
isolation of two strains of west nile virus during an outbreak in southern russia, 1999.from july to september 1999, a widespread outbreak of meningoencephalitis associated with west nile virus (flavivirus, flaviviridae) occurred in southern russia, with hundreds of cases and dozens of deaths. two strains of west nile virus isolated from patient serum and brain-tissue samples reacted in hemagglutination-inhibition and neutralization tests with patients' convalescent-phase sera and immune ascites fluid from other strains of west nile virus.200010905970
human recombinant laminin-binding protein: isolation, purification, and crystallization.the mrna of the precursor of laminin-binding protein (lbp) was isolated from a human embryo kidney cell line and cloned. the determined sequence of the lbp gene showed complete identity with the lbp genes isolated from human lung and large intestine cells. the human lbp was expressed by e. coli cells, and it was purified using ni-nta-sepharose chromatography. the mobility of the homogeneous recombinant human laminin-binding protein on sds-page was 43 kd. a mixture of eight murine monoclonal anti ...200010851030
phylogenetic comparison of the den-2 mexican isolate with other flaviviruses.recent attention has focused on the geographic variation of dengue viruses, since major epidemies may follow introduction of a new virus strain into susceptible populations. we cloned and sequenced a very interesting mexican isolate (200787/1983) which is antigenically unique by signature analysis with respect to all other dengue-2 topotype viruses. this strain is also unique in biological behavior (neurotropism) and is of epidemiological significance in mexico. the dengue-2 mexican isolate sequ ...200010773737
rna viruses: emerging vectors for vaccination and gene therapy.the application of modern molecular techniques has profoundly influenced our understanding of virus function. as a consequence, virus biology is being directly applied to medical research. it is a reflection of the current pace of virology that we are now beginning to think of our ancient foes as useful and beneficial tools.200010637572
[ocular manifestation in dengue fever].dengue-fever is a mosquito transmitted viral disease. it is endemic mainly in south-east asia and africa. the clinical manifestation is divided into different stages. ocular manifestations in travellers have been rarely reported.200010916388
mother-to-infant transmission of gb virus type c/hgv.the potentially hepatotropic flavivirus-like virus, gb virus type c (gbv-c)/hgv, has been detected in a few patients with acute and chronic hepatitis and in a certain proportion of blood donors and recipients of blood or blood components.200010864996
chimeric langat/dengue viruses protect mice from heterologous challenge with the highly virulent strains of tick-borne encephalitis virus.langat virus (lgt), a tick-borne flavivirus, is naturally attenuated for humans but it is very virulent in scid mice. in contrast, viable recombinant chimeras of lgt (prem and e genes) and dengue type 4 virus (all other sequences) recovered in mosquito cell culture were completely attenuated in scid mice but still capable of providing protection against lgt. to develop the chimeras into vaccine candidates, we adapted them to replicate efficiently in simian vero cells, a satisfactory substrate fo ...200010936085
[identification of the encephalitis equine virus, parana, brazil].in the period of 1996-1999 some virus associated with encephalitis have been reported in horses from different regions of paraná state, brazil. to identify the etiologic agent associated with this illness, mosquitoes and serum samples were collected in the endemic area.200010920444
codon-substitution models for heterogeneous selection pressure at amino acid sites.comparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. the nonsynonymous/synonymous rate ratio (omega = d(n)d(s)) is an important indicator of selective pressure at the protein level, with omega = 1 meaning neutral mutations, omega < 1 purifying selection, and omega > 1 diversifying positive selection. amino acid sites in a protein are expected to be under di ...200010790415
hepatitis c virus internal ribosome entry site (ires) stem loop iiid contains a phylogenetically conserved ggg triplet essential for translation and ires folding.the hepatitis c virus (hcv) internal ribosome entry site (ires) is a highly structured rna element that directs cap-independent translation of the viral polyprotein. morpholino antisense oligonucleotides directed towards stem loop iiid drastically reduced hcv ires activity. mutagenesis studies of this region showed that the ggg triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop iiid is essential for ires activity both in vitro and in vivo. sequence comparison sh ...200011044087
tick-borne encephalitis virus strains of western siberia.tick-borne encephalitis virus (tbev) strains were isolated from ticks in western siberia for 12 years. molecular hybridization of the 46 viral rna with the tbev cdna and oligonucleotide probes revealed differences between the siberian and far eastern strains. a comparison of the viral e gene fragment nucleotide sequence showed 89-98% homology between siberian tbev strains, whereas their similarity with strains from other populations was less than 83%. however, the viral e and ns1 glycoprotein an ...200011074120
phylogeny of the genus flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector 200010950993
recombinant plasmid expressing a truncated dengue-2 virus e protein without co-expression of prm protein induces partial protection in mice.a nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 e glycoprotein without concomitant expression of prm. the truncated e protein was properly expressed even in the absence of prm. mice inoculated intramuscularly with the recombinant plasmid containing 94% of the e gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines by their lymphoid cells when stimulated with purified dengue-2 virus. however, pro ...200011090717
genetic variation of japanese encephalitis virus in taiwan.a 280-nucleotide sequence from the capsid-premembrane (c/prem) gene region of 44 japanese encephalitis (je) virus strains isolated in taiwan from mosquitoes from 1983 to 1994, and 3 strains, (ling [1965], chang [1965], and hv1 [1958]) isolated from human brain were analyzed by direct sequencing of reverse transcription-polymerase chain reaction (rt-pcr) amplified products and compared with the corresponding sequences of reference strains. the overall sequence homology of the 47 isolates was > or ...200011220759
genetic heterogeneity and molecular epidemiology of gb virus c/hepatitis g virus in china.the inter- and intrapatient genetic variation of gb virus c (gbv-c)/hepatitis g virus (hgv) was investigated to characterize the molecular epidemiologic profile of gbv-c/ hgv infection in china, an area endemic for viral hepatitis. the intrapatient variation of hepatitis c virus (hcv) from the same patients was compared to that of gbv-c/hgv.200011100910
the brazilian flaviviruses.ten flaviviruses occur in brazil: bussuquara, cacipacoré, dengue 1, 2 and 4, iguape, ilhéus, rocio, saint louis encephalitis and yellow fever. aspects of sylvatic maintenance cycles and human diseases caused by these viruses are analyzed. large dengue outbreaks are occurring in brazil and there is a risk of yellow fever urbanization.200011113383
the rna aptamer-binding site of hepatitis c virus ns3 protease.nonstructural protein 3 (ns3) of hepatitis c virus (hcv) is a trypsin-like protease and is essential for processing of viral polyprotein. accordingly, it is a potential target for anti-hcv drugs. recently we could isolate rna aptamers (g9-i, ii, and iii) which bind and inhibit ns3 protease using in vitro selection strategy. in addition, g9-i aptamer showed noncompetitive inhibition. in order to elucidate the binding site of g9-i aptamer in ns3 protease domain (deltans3), we carried out alanine s ...200011118325
recent advances in prevention and treatment of hepatitis c virus infections.hepatitis c virus (hcv) is the leading cause of chronic hepatitis in humans. as members of the flavivirus family, hcvs are a group of small single-stranded, positive-sense rna viruses. upon translation of the genome, a polyprotein precursor is synthesized and further processed by both cellular and viral proteases to generate functional viral proteins. treatment options are currently limited to the administration of alpha-interferon alone or in combination with ribavirin. unfortunately, these app ...200011127961
[west nile virus (wnv) encephalitis].a disease on the come back: west nile encephalitis has been known since the thirties. it generally occurs in africa and western asia, rarely in europe. the disease has disappeared from france since 1960, but has reappeared in the united states and in israel. a severe viral infection: west nile encephalitis is caused by a flavivirus. after 2 to 15 days incubation, the patient experiences fever, headache, diffuse pain, and sometimes skin rash. disorientation, stiff neck, convulsions and paralysis ...200011155732
solution structure and dynamics of the single-chain hepatitis c virus ns3 protease ns4a cofactor complex.the backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis c virus ns3 protease ns4a cofactor complex have been determined by nmr spectroscopy. residues i34 to s181 of ns3 and the central three residues of the ns4a cofactor were assigned and the secondary structure was verified for these residues. in several x-ray structures of ns4a-bound ns3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the a0 strand and ...200111162117
innate resistance to flavivirus infection in mice controlled by flv is nitric oxide-independent.innate resistance to flaviviruses in mice is active in the brain where it restricts virus replication. this resistance is controlled by a single genetic locus, flv, located on mouse chromosome 5 near the locus encoding the neuronal form of nitric oxide synthase (nos1). since nitric oxide (no) has been implicated in antiviral activity, its involvement in natural resistance to flaviviruses has been hypothesized. here we present data on no production before and during flavivirus infection in both b ...200111172102
west nile virus vaccine.within the past 5 years, west nile encephalitis has emerged as an important disease of humans and horses in europe. in 1999, the disease appeared for the first time in the northeastern united states. west nile virus (a mosquito-borne flavivirus) has flourished in the north american ecosystem and is expected to expand its geographic range. in this review, the rationale for a human and veterinary vaccine is presented and a novel approach for rapid development of a molecularly-defined, live, attenu ...200112455232
solomon islands dengue seroprevalence study--previous circulation of dengue confirmed.since there were no confirmed outbreaks of dengue on record an extensive search of available records and a seroprevalence study were undertaken to determine if there was evidence of past dengue virus infections in the solomon islands. hospital and health department records revealed a suspected dengue outbreak in 1982 and deaths attributed to dengue in 1995 and 1996. serum samples from blood donors were analyzed for the presence of igg antibodies specific for flavivirus and dengue virus. a total ...200112418677
recently discovered blood-borne viruses.in recent years, molecular biology advances have enabled many investigators to discover a number of viruses that have been difficult to characterise by cell culture techniques. two blood-borne viruses have been identified. these are gb virus c (gbv-c) and tt virus (ttv). gbv-c was discovered in 1995. it is a flavivirus-like enveloped particle measuring 50-100 nm in diameter with a density of 1.08-1.13 g/cm3. the genome of gbv-c is a single-stranded, positive strand ribonucleic acid of approximat ...200111948357
iminosugars as possible broad spectrum anti hepatitis virus agents: the glucovirs and alkovirs.glucosidases in the endoplasmic reticulum (er) mediate the first step in processing n-linked oligosaccharides. recent evidence suggests that morphogenesis and secretion of members of the hepatitis b and flavivirus families are more dependent on these enzymes than are most host glycoproteins. thus, it is possible that glucosidase inhibitors can be designed that are safe and selective for the treatment of hepatitis b and possibly c (since hepatitis c virus is a member of the flavivirus family), ma ...200112018676
phylogenetic and evolutionary relationships among yellow fever virus isolates in africa.previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (yf) virus in africa, one in west africa and the other in east and central africa. we have examined the prm/m and a portion of the e protein for a panel of 38 wild strains of yf virus from africa representing different countries and times of isolation. examination of the strains revealed a more complex genetic relationship than previously reported. overall, nucleotide substitutions varie ...200111435580
flaviviruses isolated from mosquitoes collected during the first recorded outbreak of japanese encephalitis virus on cape york peninsula, australia.in response to an outbreak of japanese encephalitis (je) virus on cape york peninsula, australia, in 1998, mosquitoes were collected using co2 and octenol-baited centers for disease control and prevention light traps. a total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. no isolates of je virus were recovered from these mosquitoes. however, 18 isolates of kokobera virus, another flavivirus were obtained from culex annulirostris. twelve isolates were from ...200111442206
[synthesis, immunogenicity and antigenic characteristics of the glycoprotein e fragments from the tick-borne encephalitis virus].six peptide fragments of the envelope protein e of the tick-borne encephalitis virus involving the predicted t-helper epitopes were synthesized. their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. the peptide binding to the antiviral antibodies was studied, and two peptides were revealed tha ...200111443938
amino acid substitution(s) in the stem-anchor region of langat virus envelope protein attenuates mouse neurovirulence.the identification of variants that are unable to bind membrane receptor preparations (mrps) has previously been shown to select attenuated yellow fever and japanese encephalitis viruses. in this study, this methodology has been extended to the tick-borne serocomplex of flaviviruses. langat (lgt) virus strain tp21 was bound to mouse or human brain mrps and viruses that escaped binding were isolated and characterized. in addition, variant viruses escaping neutralization by the monoclonal antibody ...200111448158
role of metastability and acidic ph in membrane fusion by tick-borne encephalitis virus.the envelope protein e of the flavivirus tick-borne encephalitis (tbe) virus is, like the alphavirus e1 protein, a class ii viral fusion protein that differs structurally and probably mechanistically from class i viral fusion proteins. the surface of the native tbe virion is covered by an icosahedrally symmetrical network of e homodimers, which mediate low-ph-induced fusion in endosomes. at the ph of fusion, the e homodimers are irreversibly converted to a homotrimeric form, which we have found ...200111462011
monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells.the specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. to study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (den) virus e-glycoprotein-specific monoclonal antibodies (mabs) to block virus adsorption to vero cells. in contrast to previous studies, the binding sites of these mabs were localized to one of three structural domains (i, ii, and iii) in the e glycoprotein. the results indicate that m ...200111462053
the relationships between west nile and kunjin viruses.until recently, west nile (wn) and kunjin (kun) viruses were classified as distinct types in the flavivirus genus. however, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. to better define this relationship, we performed sequence analyses on 32 isolates of kun virus and 28 isolates of wn virus from different geographic areas, including a wn isolate from the recent outbreak in new york. sequence comparisons showed that th ...200111585535
molecular organization of a recombinant subviral particle from tick-borne encephalitis virus.the tick-borne encephalitis (tbe) flavivirus contains two transmembrane proteins, e and m. coexpression of e and the m precursor (prm) leads to secretion of recombinant subviral particles (rsps). in the most common form of these rsps, analyzed at a 19 a resolution by cryo-electron microscopy (cryo-em), 60 copies of e pack as dimers in a t = 1 icosahedral surface lattice (outer diameter, 315 a). fitting the high-resolution structure of a soluble e fragment into the rsp density defines interaction ...200111463384
pathologic and immunohistochemical findings in naturally occuring west nile virus infection in horses.the pathologic and peroxidase immunohistochemical features of west nile flavivirus (wnv) infection were compared in four horses from the northeastern united states and six horses from central italy. in all 10 animals, there were mild to severe polioencephalomyelitis with small t lymphocyte and lesser macrophage perivascular infiltrate, multifocal glial nodules, neutrophils, and occasional neuronophagia. perivascular hemorrhages, also noted macroscopically in two animals, were observed in 50% of ...200111467475
salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa b activation.flaviviruses comprise a positive-sense rna genome that replicates exclusively in the cytoplasm of infected cells. whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. flavivirus infections quickly activate nuclear factor kappa b (nf-kappab), and salicylates have been shown to inhibit nf-kappab activation. in this study, we investigated whether salicylates suppress flavivirus replication and virus-induced ...200111483726
tick-borne langat/mosquito-borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: evaluation in rhesus monkeys and in mosquitoes.langat virus (lgt), strain tp21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained lgt genes for premembrane (prem) and envelope (e) glycoprotein and all other sequences derived from dengue type 4 virus (den4). the live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective e epitopes among themselves and with lgt. toward that end the chimera, initi ...200111483771
assessment of substrate specificity of hepatitis g virus ns3 protease by a genetic method.the rna genome of hepatitis g virus (hgv) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. the hgv ns3 protease is responsible for the cleavage of the hgv polyprotein at four different locations. no conserved sequence motif has been identified for the cleavage sites of the ns3 protease. to determine the substrate specificity of the ns3 protease, amino acid sequences cleaved by the ns3 protease were obtained from randomized sequence libraries by using a ...200111485324
distribution and characterization of tick-borne encephalitis viruses from siberia and far-eastern asia.in this study, tick-borne encephalitis (tbe) viruses from siberia and far-eastern asia were characterized in order to determine virus subtype distribution. tbe viruses were isolated from ticks (ixodes persulcatus) collected in the far-eastern (khabarovsk and vladivostok) and siberian (irkutsk) regions of russia in 1999. phylogenetic analysis showed that isolates formed distinct clusters of far-eastern and siberian subtypes. there was also a minor difference in antigenicity between the irkutsk is ...200111369875
infection with langat flavivirus or expression of the envelope protein induces apoptotic cell death.langat (lgt) flavivirus, derived from infectious full-length cdna clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (neuro-2a) and simian kidney (vero and llc-mk(2)) cells. the hallmark of apoptosis, cleavage of cellular dna, was observed 48 h after infection of vero, llc-mk(2), and neuro-2a cells by electrophoresis analysis. apoptosis in infected cells was also confirmed by tunel assay. lgt-infected neuro-2a cells showed an increase in caspase-3-like protease (devd ...200111485400
rapid elimination of gb virus c (hepatitis g virus) in the mosquito aedes aegypti.the transmissibility of the gb virus c (hepatitis g virus; hgv), a member of the flaviviridae, by a typical flavivirus vector was investigated. female mosquitoes of the species aedes aegypti were fed with hgv-infected human blood and assayed 1, 24, 48, 72 and 96 h after the blood meal for viral rna, human glyceraldehyde-3-phosphate dehydrogenase mrna, human beta-actin dna and a. aegypti actin mrna by total nucleic acid extraction, reverse transcription and pcr. viral rna had already disappeared ...200111489416
[pathogenesis of hemorrhagic fever]. 200111496775
terminal nucleotidyl transferase activity of recombinant flaviviridae rna-dependent rna polymerases: implication for viral rna synthesis.recombinant hepatitis c virus (hcv) rna-dependent rna polymerase (rdrp) was reported to possess terminal transferase (tntase) activity, the ability to add nontemplated nucleotides to the 3' end of viral rnas. however, this tntase was later purported to be a cellular enzyme copurifying with the hcv rdrp. in this report, we present evidence that tntase activity is an inherent function of hcv and bovine viral diarrhea virus rdrps highly purified from both prokaryotic and eukaryotic cells. a change ...200111507207
determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein ns1 of murray valley encephalitis virus.the 12 cysteine residues in the flavivirus ns1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. in this study, we examined the intramolecular disulfide bond arrangement of ns1 of murray valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. disulfide linkages were identified by separating tryptic-digested ns1 by reverse-phase high pressure liquid chromatography and an ...200111514736
dengue and other emerging flaviviruses.flaviviruses are among the most important emerging viruses known to man. most are arboviruses (arthropod-borne) being transmitted by mosquitoes or ticks. they derived from a common ancestor 10-20000 years ago and are evolving rapidly to fill new ecological niches. many are spreading to new geographical areas and causing increased numbers of infections. traditionally, three clinical syndromes are recognized: fever-arthralgia-rash, viral haemorrhagic fever, and neurological disease, though for som ...200111531316
de novo synthesis of rna by the dengue virus rna-dependent rna polymerase exhibits temperature dependence at the initiation but not elongation phase.replication of positive strand flaviviruses is mediated by the viral rna-dependent rna polymerases (rdrp). to study replication of dengue virus (den), a flavivirus family member, an in vitro rdrp assay was established using cytoplasmic extracts of den-infected mosquito cells and viral subgenomic rna templates containing 5'- and 3'-terminal regions (trs). evidence supported that an interaction between the trs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements i ...200111546770
recent advances in prevention and treatment of hepatitis c virus infections.hepatitis c virus (hcv) is the leading cause of chronic hepatitis in humans. as members of the flavivirus family, hcvs are a group of small single-stranded, positive-sense rna viruses. upon translation of the genome, a polyprotein precursor is synthesized and further processed by both cellular and viral proteases to generate functional viral proteins. treatment options are currently limited to the administration of alpha-interferon alone or in combination with ribavirin. unfortunately, these app ...200111548211
construction, characterization and immunogenicity of recombinant yellow fever 17d-dengue type 2 viruses.chimeric yellow fever (yf)-dengue type 2 (den 2) viruses were constructed by replacing the premembrane (prm) and envelope (e) genes of yf 17d virus with those from den 2 virus strains of south-east asian genotype. whereas viable chimeric viruses were successfully recovered when the yf 17d c gene and the den 2 prm gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. unlike yf virus that replicated in all the ce ...200111551641
nucleotide sequencing and serological evidence that the recently recognized deer tick virus is a genotype of powassan virus.deer tick virus (dtv) is a recently recognized north american virus isolated from ixodes dammini ticks. nucleotide sequencing of fragments of structural and non-structural protein genes suggested that this virus was most closely related to the tick-borne flavivirus powassan (pow), which causes potentially fatal encephalitis in humans. to determine whether dtv represents a new and distinct member of the flavivirus genus of the family flaviviridae, we sequenced the structural protein genes and 5' ...200111551648
complete coding sequence of the alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in saudi arabia.to date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in siberia (omsk hemorrhagic fever virus), india (kyasanur forest disease virus, kfdv), and in saudi arabia (alkhurma virus, alkv). prior to this study, only partial coding sequences of these severe pathogens had been determined. we report here the complete coding sequence of alk virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. indepe ...200111554750
mutagenesis of the dengue virus type 2 ns3 protein within and outside helicase motifs: effects on enzyme activity and virus replication.the protein ns3 of dengue virus type 2 (den-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the n-terminal region and an ntpase-helicase in the remaining 70% of the protein. the latter region has seven conserved helicase motifs found in all members of the family flaviviridae. den-2 ns3 lacking the proteinase region was synthesized as a fusion protein with glutathione s-transferas ...200111559795
chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in vero cells or human liver cells and attenuated in mice.a recombinant live attenuated dengue virus type 4 (den4) vaccine candidate, 2adelta30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2adelta30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (delta30) in the 3' untranslated region (utr) of the viral genome. in the present study, chemical mutagenesis of den4 was utilized to generate attenuating mutations which may ...200111559806
transcriptional regulation of major histocompatibility complex class i by flavivirus west nile is dependent on nf-kappab activation.infection by the flavivirus west nile (wnv) is associated with a virus-specific increase of major histocompatibility complex class i (mhc-i) molecules on the cell surface of diploid vertebrate cells. the increased mhc-i cell surface expression is functional and is associated with increased susceptibility to secondary wnv-immune and alloimmune cytotoxic t cells. wnv-induced up-regulation of cell surface mhc-i expression is associated with nf-kappab activation and increased transcription of mhc-i ...200111574908
phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography.phylogenetic analysis of the flavivirus genus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. these phylogenies identified mosquito-borne, tick-borne and no-known-vector (nkv) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. the mosquito-borne flaviviruses revealed two distinct epidemiological groups: ( ...200111457992
activity of recombinant dengue 2 virus ns3 protease in the presence of a truncated ns2b co-factor, small peptide substrates, and inhibitors.recombinant forms of the dengue 2 virus ns3 protease linked to a 40-residue co-factor, corresponding to part of ns2b, have been expressed in escherichia coli and shown to be active against para-nitroanilide substrates comprising the p6-p1 residues of four substrate cleavage sequences. the enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. the ns4b/n ...200111581268
isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein ns4b in viral cytopathogenicity.isolates of bovine viral diarrhea virus (bvdv), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. the cp viruses also differ from ncp viruses by the production of viral nonstructural protein ns3. however, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. a bicistronic b ...200111602707
mutations in the ns3 gene and 3'-ncr of japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus.the t1p1 strain of japanese encephalitis (je) virus was recently isolated from paddy-free liu-chiu islet in which natural je antibody has been prevalent. in mouse neuroblastoma-derived neuro-2a cells, t1p1 appeared significantly lower in virus productivity than another local isolate, ch1392. it implied that this new isolate possesses a characteristic viral replication pattern other than that of ch1392. t1p1 has also shown lower neurovirulence, which was reflected by a significantly higher ld(50) ...200111601924
the west nile virus encephalitis outbreak in the united states (1999-2000): from flushing, new york, to beyond its borders.viruses cause most forms of encephalitis. the two main types responsible for epidemic encephalitis are enteroviruses and arboviruses. the city of new york reports about 10 cases of encephalitis yearly. establishing a diagnosis is often difficult. in august 1999, a cluster of five patients with fever, confusion, and weakness were admitted to a community hospital in flushing, new york. flaccid paralysis developed in four of the five patients, and they required ventilatory support. three, less seve ...200111797774
a phylogenetic approach to following west nile virus in connecticut.the 1999 outbreak of west nile (wn) virus in the northeastern united states was the first known natural occurrence of this flavivirus in the western hemisphere. in 1999 and 2000, 82 independent connecticut wn virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with wn-ny99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ...200111606791
immunization of mice against west nile virus with recombinant envelope protein.west nile (wn) virus is a mosquito-borne flavivirus that emerged in the united states in 1999 and can cause fatal encephalitis. envelope (e) protein cdna from a wn virus isolate recovered from culex pipiens in connecticut was expressed in escherichia coli. the recombinant e protein was purified and used as ag in immunoblot assays and immunization experiments. patients with wn virus infection had abs that recognized the recombinant e protein. c3h/hen mice immunized with e protein developed e prot ...200111673542
a novel mechanism to explain protein abnormalities in schizophrenia based on the flavivirus resistance gene.the geographical distribution of schizophrenia was previously shown to correlate with the global distribution of tick-borne flaviviruses. the correlation suggests a natural resistance gene to flaviviruses could be involved in schizophrenia. the flavivirus resistance gene, flv, a gene found in wild mice and certain in-bred strains, confers resistance to flaviviruses through the interaction of cellular proteins with the flaviviral 3' untranslated regions (utrs). although the sequence and product o ...200111673799
evolution, epidemiology, and dispersal of flaviviruses revealed by molecular phylogenies. 200111680389
sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.sequences of 191 flavivirus rnas belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' ncr) directly upstream of the conserved terminal hairpin. in mosquito-borne flavivirus rnas (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. this element is repeated in the dengue and japanese encephalitis rnas and centers around the conserved sequences cs2 and rcs2. in yellow fever virus ...200111680841
the machinery for flavivirus fusion with host cell membranes.a combination of structural, biochemical and functional studies with the flavivirus tick-borne encephalitis virus has revealed the characteristics of a new class of viral fusion protein, class ii, that is unrelated to the class i viral fusion proteins for which influenza virus hemagglutinin is the prototype. new structural data have shown that the alphaviruses, another group of icosahedral enveloped viruses, also have class ii fusion proteins, suggesting a common origin.200111495810
experimental infections of pigs with japanese encephalitis virus and closely related australian flaviviruses.the flavivirus japanese encephalitis (je) virus has recently emerged in the australasian region. to investigate the involvement of infections with related enzootic flaviviruses, namely murray valley encephalitis (mve) virus and kunjin (kun) virus, on immunity of pigs to je virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. antibody responses to primary and secondary infections were evaluated using panels o ...200111693888
role of reactive oxygen intermediates in japanese encephalitis virus infection in murine neuroblastoma cells.infection with japanese encephalitis virus (jev), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. this study attempted to determine whether jev infection induces free radical generation and whether oxidative stress contributes to virus-induced cell death in neuroblastoma cells. a rise in the intracellular level of free radicals indicated by the 2',7'-dichlorofluorescein fluorescence was obse ...200111711202
nucleic acid sequence-based amplification assays for rapid detection of west nile and st. louis encephalitis viruses.the development and application of nucleic acid sequence-based amplification (nasba) assays for the detection of west nile (wn) and st. louis encephalitis (sle) viruses are reported. two unique detection formats were developed for the nasba assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (nasba-ecl assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (nasba-beacon assay). the sen ...200111724870
characterization of tick-borne encephalitis virus from latvia: evidence for co-circulation of three distinct subtypes.viruses of the tick-borne encephalitis (tbe) antigenic complex within the family flaviviridae cause a variety of diseases, including uncomplicated febrile illness, meningoencephalitis, and hemorrhagic fever. different domesticated animals or wildlife species often act as reservoir hosts and ixodid ticks serve as vectors. although tbe is a serious problem in latvia, the knowledge concerning tbe virus (tbev) strains circulating in the country is most limited. only two strains (latvia-1-96 isolated ...200111745938
transport and budding at two distinct sites of visible nucleocapsids of west nile (sarafend) virus.it has been difficult to detect and visualize the physical nucleocapsid particles during the replication process of the flaviviruses. the use of cryo-immunoelectron microscopy has clearly revealed the capsid proteins and nucleocapsid particles of west nile (sarafend) virus (a flavivirus) for the first time. physical nucleocapsid particles accumulated in large numbers from 8 hr postinfection. double immunolabeling of the envelope and capsid proteins showed a close association of these structural ...200111745942
effector cytolotic function but not ifn-gamma production in cytotoxic t cells triggered by virus-infected target cells in vitro.cytolysis and interferon(ifn)-gamma production are two independent effector functions of activated cytotoxic t (tc) cells. we have used the tc-cell response against the flavivirus, murray valley encephalitis virus (mve), to investigate the requirements for inducing these two functions with regard to antigen-concentration and cd8 coreceptor involvement. cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo mve-immune as well as in vitro peptide-restimulated splenocytes at low ...200111555403
experimental yellow fever virus infection in the golden hamster (mesocricetus auratus). i. virologic, biochemical, and immunologic studies.this report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (yf) virus. an accompanying paper describes the pathologic findings. following intraperitoneal inoculation of a virulent strain of yf virus, hamsters developed a high-titered viremia (up to 109/ml) lasting 5--6 days and abnormal liver function tests. yf hemagglutination-inhibiting antibodies appeared 4 or 5 days after infection, often while viremia was still present. the mortality ...200111319679
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