Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.
Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. (+info)
Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein.
T cells must play the major role in controlling acute human Lassa virus infection, because patients recover from acute Lassa fever in the absence of a measurable neutralizing antibody response. T cells alone seem to protect animals from a lethal Lassa virus challenge, because after experimental vaccination no neutralizing antibodies are detectable. In order to study human T-cell reactivity to single Lassa virus proteins, the nucleoprotein (NP) of Lassa virus, strain Josiah, was cloned, expressed in Escherichia coli, and affinity purified. Peripheral blood mononuclear cells (PBMC) obtained from 8 of 13 healthy, Lassa virus antibody-positive individuals living in the Republic of Guinea, western Africa, were found to proliferate in response to the recombinant protein (proliferation index >/=10). PBMC obtained from one individual with a particularly high proliferative response were used to generate 50 NP-specific T-cell clones (TCC). For six of these the epitopes were mapped with overlapping synthetic peptides derived from the sequence of the NP. These CD4(+) TCC displayed high specific proliferation and produced mainly gamma interferon upon stimulation with NP. Because variation of up to 15% in the amino acid sequences of the structural proteins of naturally occurring Lassa virus variants has been observed, the reactivity of the TCC with peptides derived from the homologous epitopes of the Nigeria strain of Lassa virus and of the eastern Africa arenavirus Mopeia was tested. With the Nigeria strain of Lassa virus the levels of homology were 100% for two of these epitopes and 85% for three of them, whereas homology with the respective Mopeia epitopes ranged from 92 to 69%. Reactivity of the TCC with peptides derived from the variable epitopes of the Nigeria strain and of Mopeia was reduced or completely abolished. This report shows for the first time that seropositive individuals from areas of endemicity have very strong memory CD4(+) T-cell responses against the NP of Lassa virus, which are partly strain specific and partly cross-reactive with other Lassa virus strains. Our findings may have important implications for the strategy of designing recombinant vaccines against this mainly T-cell-controlled human arenavirus infection. (+info)
Diagnosis and clinical virology of Lassa fever as evaluated by enzyme-linked immunosorbent assay, indirect fluorescent-antibody test, and virus isolation.
The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the "gold standard." Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever. (+info)
Effective vaccine for lassa fever.
Lassa fever has been estimated to cause 5,000 deaths annually in West Africa. Recently, war in the zone where Lassa fever is hyperendemic has severely impeded control and treatment. Vaccination is the most viable control measure. There is no correlation between antibody levels and outcome in human patients, and inactivated vaccines produce high titers of antibodies to all viral proteins but do not prevent virus replication and death in nonhuman primates. Accordingly, we vaccinated 44 macaques with vaccinia virus-expressed Lassa virus structural proteins separately and in combination, with the object of inducing a predominantly TH1-type immune response. Following Lassa virus challenge, all unvaccinated animals died (0% survival). Nine of 10 animals vaccinated with all proteins survived (90% survival). Although no animals that received full-length glycoprotein alone had a high titer of antibody, 17 of 19 survived challenge (88%). In contrast, all animals vaccinated with nucleoprotein developed high titers of antibody but 12 of 15 died (20% survival). All animals vaccinated with single glycoproteins, G1 or G2, died, but all those that received both single glycoproteins (G1 plus G2) at separate sites survived, showing that both glycoproteins are independently important in protection. Neither group had demonstrable antibody levels prior to challenge. We demonstrate that in primates, immune responses to epitopes on both glycoproteins are required to protect against lethal challenge with Lassa virus without having untoward side effects and that this protection is likely to be primarily cell mediated. We show that an effective, safe vaccine against Lassa virus can and should be made and that its evaluation for human populations is a matter of humanitarian priority. (+info)
Genetic diversity among Lassa virus strains.
The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation. (+info)
Imported lassa fever in Germany: molecular characterization of a new lassa virus strain.
We describe the isolation and characterization of a new Lassa virus strain imported into Germany by a traveler who had visited Ghana, Cote D'Ivoire, and Burkina Faso. This strain, designated "AV," originated from a region in West Africa where Lassa fever has not been reported. Viral S RNA isolated from the patient's serum was amplified and sequenced. A long-range reverse transcription polymerase chain reaction allowed amplification of the full-length (3.4 kb) S RNA. The coding sequences of strain AV differed from those of all known Lassa prototype strains (Josiah, Nigeria, and LP) by approximately 20%, mainly at third codon positions. Phylogenetically, strain AV appears to be most closely related to strain Josiah from Sierra Leone. Lassa viruses comprise a group of genetically highly diverse strains, which has implications for vaccine development. The new method for full-length S RNA amplification may facilitate identification and molecular analysis of new arenaviruses or arenavirus strains. (+info)
Low levels of interleukin-8 and interferon-inducible protein-10 in serum are associated with fatal infections in acute Lassa fever.
To investigate the role of inflammatory mediators in the pathogenesis of Lassa fever, the levels of a number of pro- and anti-inflammatory cytokines and chemokines in serum samples collected from hospitalized patients with fatal and nonfatal acute Lassa fever were compared with those from 2 control groups: patients with other febrile illnesses and uninfected individuals. Serum interleukin (IL)-8 and interferon (IFN)-inducible protein (IP)-10 levels were significantly higher in patients with acute nonfatal Lassa fever than in control subjects. In striking contrast, levels of these chemokines were low or undetectable in patients with fatal Lassa fever. IFN-gamma, IL-12, IL-6, and RANTES levels were elevated in all the febrile study groups. Tumor necrosis factor-alpha levels were not elevated in patients with fatal or nonfatal Lassa fever. These data indicate that acute nonfatal Lassa fever is associated with high levels of circulating IL-8 and IP-10 and that low levels or absence of these mediators correlates with a poor outcome. (+info)
Mucosal immunization with Salmonella typhimurium expressing Lassa virus nucleocapsid protein cross-protects mice from lethal challenge with lymphocytic choriomeningitis virus.
OBJECTIVES: Lassa fever virus (LAS) is transmitted to man by rodent carriers and is fatal in a third of untreated cases. Our goal is to provide immune protection from Lassa fever by mucosal vaccination. STUDY DESIGN/METHODS: Mice were vaccinated intragastrically with control vectors or with vectors (vaccinia or Salmonella) expressing LAS nucleocapsid protein (NP). Mice were challenged intracranially with a lethal dose of the related arenavirus, lymphocytic choriomeningitis virus (LCMV), as a measure of the vaccine's ability to elicit cross-protection. RESULTS: Salmonella and vaccinia vectors expressing LAS NP each protected a third of the mice from lethal challenge with LCMV. All mice vaccinated with a vector expressing LCMV NP were protected as expected. CONCLUSIONS: The LAS recombinant Salmonella vector is comparable to the LAS recombinant vaccinia vector in its ability to cross-protect mice from lethal challenge. Nucleocapsid protein is an inadequate immunogen on its own, but provides sufficient cross-protection to make it a useful component of a broadly reactive arenavirus vaccine. (+info)