LCMV-mediated hepatitis in rhesus macaques: WE but not ARM strain activates hepatocytes and induces liver regeneration.

Lymphocytic chorimeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), causative agent of Lassa hemorrhagic fever (LHF), belong to the Old World group of the family Arenaviridae. Both viruses have extensive strain diversity and significant variations in lethality and pathogenicity for man and experimental animals. We have shown that the LHF-like infection of rhesus macaques with the WE strain of LCMV affects liver functions, induces hepatocyte proliferation, and causes a rise in IL-6 and soluble TNF receptors (sTNFR) concomitant with a rise in viremia. The levels of IL-6 and sTNFR can serve as an additional diagnostic tool for liver involvement in pathogenesis of arenavirus infection. Mucosal inoculation of rhesus macaques with LCMV-WE can result in attenuated infection with a transient viremia and liver enzyme abnormalities. The ARM strain of LCMV shares 88% amino acid homology with WE. In contrast to LCMV-WE, ARM strain does not induce manifested disease in monkeys, does not affect liver functions, and does not induce hepatocyte proliferation. Previously we demonstrated that LCMV-ARM infection protected rhesus macaques challenged with LCMV-WE. Here we have shown that the protected animals have no signs of hepatitis and hepatocyte proliferation.

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.

Role of the promyelocytic leukemia protein PML in the interferon sensitivity of lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) induces type I interferon (alpha and beta interferon [IFN-alpha and IFN-beta]) upon infection and yet is sensitive to the addition of type II interferon (gamma interferon [IFN-gamma]) to the culture media. This sensitivity is biologically important because it correlates inversely with the ability of certain LCMV strains to persist in mice (D. Moskophidis, M. Battegay, M. A. Bruendler, E. Laine, I. Gresser, and R. M. Zinkernagel, J. Virol. 68:1951-1955, 1994). The cellular oncoprotein PML is induced by both IFN-alpha/beta and IFN-gamma, and PML binds the LCMV Z protein and becomes redistributed within cells from nucleus to cytoplasm upon LCMV infection. In the present study, increased PML expression results in diminished LCMV replication, implicating PML in the IFN sensitivity of LCMV. Virus production in PML -/- murine embryonic fibroblasts (MEF) exceeds virus production in PML +/+ MEF, and this difference is exacerbated by IFN treatment that upregulates PML expression. IFN-gamma also diminishes LCMV production in PML -/- cells; therefore, viral IFN sensitivity is not entirely due to PML. Both viral mRNA production and viral protein production decrease as PML expression increases. Here we propose that PML reduces LCMV transcription through its interaction with the Z protein.

Murine immune responses to mucosally delivered Salmonella expressing Lassa fever virus nucleoprotein.

Arenaviruses are emerging pathogens known to infect via the mucosa, however no formal attempts to make mucosal vaccines have been undertaken. Here we describe a recombinant aroA attenuated Salmonella typhimurium that expresses the nucleoprotein (NP) gene of Lassa fever virus (LAS). The complete NP gene was cloned downstream of the bacterial groEL promotor and integrated into the aroA locus of S. typhimurium. Lassa NP protein was detected in whole cell extracts from the recombinant Salmonella by immunoblot analysis with serum from Lassa-infected people. Mice were inoculated by intragastric intubation with 5 x 10(9) S. typhimurium and boosted with the same recombinant Salmonella 21 days after the primary inoculation. Both local mucosal IgA and serum immunoglobulins against Lassa NP were observed. Splenic cytotoxic T-lymphocyte responses to LAS NP were detected after the boost and they cross-reacted with target cells infected with the related arenavirus, lymphocytic choriomeningitis virus. Recombinant Salmonella elicits humoral and cell mediated immune responses against Lassa fever virus in mice and should be considered as a potential vaccine strategy in man.

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.

Sequence comparison of the large genomic RNA segments of two strains of lymphocytic choriomeningitis virus differing in pathogenic potential for guinea pigs.

Two strains of lymphocytic choriomeningitis virus (LCMV) differ in their ability to cause a lethal disease in outbred guinea pigs: the Armstrong (ARM) strain is not lethal at high doses (10(6) PFU), whereas the WE strain is lethal at less than 10 PFU inoculated intraperitoneally. The high pathogenic potential of LCMV WE has been mapped to the larger (L) of the two genomic RNA segments by genetic reassortment analysis (Riviere, Y., Ahmed, R., Southern, P. J., Buchmeier, M. J. and Oldstone, M. B. A., J. Virol. 55, 704-709, 1985). Here we describe the completed sequence of the LCMV WE L RNA, and its comparison to the L RNA of the non-virulent strain, LCMV ARM. Similar to the L RNA of LCMV ARM, the L RNA of WE is 7.2 kb long and contains two open reading frames (ORFs): the 5" ORF encodes a small RING finger (zinc-binding) protein, p11 Z, and the 3" ORF encodes the putative RNA-dependent RNA polymerase (RdRp or L protein). Comparison of nucleotide sequences for both viruses revealed 84% L RNA homology. At the amino acid level similarity between the two strains is 87% in the Z ORF, and 88% in the RdRp ORF. The most divergent regions are found in the N-terminal parts of the RdRp and Z proteins and are most likely to account for differences in pathogenic potential.

Completion of the Lassa fever virus sequence and identification of a RING finger open reading frame at the L RNA 5' End.

Lassa (LAS) fever virus is a highly pathogenic arenavirus with large (L) and small (S) RNA genomic segments. The 5' end of the LAS L segment is described here, thereby completing the sequence of the most virulent arenavirus analyzed to date. In keeping with the ambisense gene structure of the arenaviruses, the LAS L RNA encodes a 250-kDa protein and an 11-kDa protein in opposite senses with respect to each other. The 11-kDa protein, defined previously in arenaviruses lymphocytic choriomeningitis (LCM), Tacaribe (TAC), and Pichinde (PIC), contains a RING type of zinc-binding structure. Expression of the 11-kDa protein in LAS virus-infected cells has been confirmed by binding to peptide-specific antibody.

The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum.

The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.

Lassa virus activity in Guinea: distribution of human antiviral antibody defined using enzyme-linked immunosorbent assay with recombinant antigen.

More than 3,100 households in 27 selected villages distributed in the main geographic regions of Guinea were surveyed for the presence of Lassa virus-specific IgG antibodies (LVA), using an enzyme-linked immunosorbent assay (ELISA) with Lassa virus nucleocapsid protein expressed in insect cells infected with a recombinant baculovirus as antigen. The highest prevalence of LVA (25-55%) was found among inhabitants of tropical secondary forest (areas near Gueckedou, Yomou, and Lola) and guinea savannah (Faranah and Kindia areas), near the southern frontiers with Sierra Leone and Liberia. A much lower prevalence (4-7%) was found among inhabitants of mountainous (Pita, Labe, and Mali) and coastal (Boffa, Boké) areas. We found no discernible differences in LVA prevalence between males and females or among various age groups. Testing of 406 hospital staff members of the eight central hospitals in these areas for LVA revealed a similar distribution of seropositivity among hospitals in various prefectures. The highest prevalence of LVA in hospital staff (29-40%) was in the Gueckedou and Lola hospitals. Sera of LVA-positive persons were tested via Western blot analysis. Antibodies bound predominantly to NP and GP2 proteins.

[The penetration of the Marburg virus into eukaryotic cells]. / Proniknovenie virusa Marburg v éukarioticheskie kletki.

The early stages of infection of Vero-E6 cell culture with Marburg virus, a member of filovirus family, highly pathogenic for man, were studied. Virus multiplication was completely or significantly inhibited by lysosomotropic agents (LTA) of two types: weak base (ammonium chloride) and ionophore monensin. The level of the inhibiting effect was proportional to LTA concentration and was maximal when the drug was introduced into the culture medium before virus inoculation. Complete inhibition of Marburg virus replication in Vero-E6 cells in the presence of 20 (30) mM ammonium chloride ("lysosomotropic blocking") was overcome by a short-time treatment of the cell culture with the virus adsorbed on it using a medium with a weak-acid pH (4.0-5.0). The results are discussed from the point of view of the mode of this virus penetration into eukaryotic cells.

[Serological evidence of Lassa virus circulation in the Republic of Guinea]. / Serologicheskie dokazatel'stva tsirkuliatsii virusa Lassa v Gvineiskoi Respublike.

Seroepidemiological investigations in Guinea were carried out to estimate the areas of Lassa virus circulation. The recombinant protein of Lassa virus nucleocapsid was used as the antigen to analyse blood sera by ELISA. In some regions, from 30 to 54.9% of the population had antibodies to Lassa virus, but in others only 6-7%.

[The localization and preliminary characteristics of antigenic sites (B epitopes) in the nucleocapsid protein of the Lassa virus]. / Lokalizatsiia i predvaritel'naia kharakteristika antigennykh saitov (B-épitopov) v belke nukleokapsida virusa Lassa.

Nucleocapsid protein of Lassa virus contains 4 epitope sites. Three of them were localized in amino acid position 123-1 27, 337-346, and 518-527. Monoclonal antibody 1108 specific to Lassa virus NP-protein reacted with 123-127 synthetic peptide in solid-phase ELISA and precipitated nucleocapsid proteins of Machupo, Tacaribe, and Mopeia arenaviruses in RIP.

Generation of reassortants between African arenaviruses.

Lassa (LAS) and Mopeia (MOP) viruses are African arenaviruses which are carried by wild rodents and occasionally transferred to humans. In humans and nonhuman primates, Lassa causes mortality in 60% of untreated cases, whereas Mopeia does not cause mortality and has been known to protect monkeys from lethal challenge with Lassa. These two African arenaviruses also differ in their lethality for suckling outbred mice and in their plaque sizes under agar overlay. MOP virus induces small plaques and lethal infection after intracerebral (ic) inoculation. In contrast, LAS inoculation does not kill mice and the virus induces large plaques. After coinfection of Vero cells with LAS and MOP viruses some phenotypic reassortants which produced small plaques and were not lethal for outbred mice were isolated and plaque-purified. Dot-blot hybridization using LAS and MOP cDNA probes specific for L and S RNA segments revealed a genotype consisting of the L RNA of MOP and the S RNA of LAS (MOP/LAS reassortant). Adoptive transfer experiments demonstrated an ability of immune splenocytes from CBA mice intraperitoneally infected with the MOP/LAS reassortants to protect recipient mice against lethal disease after ic inoculation with LAS virus.

[The detection of the Marburg virus antigen by solid-phase immunoenzyme analysis]. / Vyiavlenie antigena virusa Marburg metodom tverdofaznogo immunofermentnogo analiza.

Comparative studies of two variants of the enzyme-linked immunosorbent assay (ELISA) were carried out to determine the sensitivity of the detection of Marburg virus antigens in Vero cells. Both competitive and two-antibody ELISA variants detected as little as 5 ng of Marburg virus antigen. The Vero cell monolayer was found to produce 5-50 ng/0.05 ml of the virus-specific proteins at 6 to 8 days postinfection.

[Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses]. / Sliianie iskusstvennykh lipidnykh membran indutsiruemoe sinteticheskim "peptidom sliianiia" arenavirusov.

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.

[The isolation and characteristics of reassortants between the Lassa and Mopeia arenaviruses]. / Poluchenie i kharakteristika reassortantov mezhdu arenavirusami Lassa i Mopeiia.

Reassortants with a mixed phenotype were produced by combined inoculation of Vero cells with Lassa and Mopeya viruses. These reassortants produced small plaques (Mopeya virus phenotype) and were not pathogenic for newborn mice (Lassa virus phenotype). The genotype of the reassortants was studied by dot hybridization experiments on filters using cDNA-probes differentiating genome segments of these viruses. The reassortants were shown to have Mopeya virus L-RNA and Lassa virus S-RNA.

[Recombinant monoclonal antibodies to the Lassa virus: the formation of reactive paratopes]. / Rekombinantnye monoklonal'nye antitela k virusu Lassa: formirovanie reaktivnykh paratopov.

Eighteen hybrid lines secreting recombinant monoclonal antibodies to Lassa virus were produced by fusion of mouse splenocytes with antibody-secreting X-63 myeloma cells. Interrelations between the structure and reactivity of the antibodies were studied by different serological and immunochemical methods. Monoclonal antibodies were divided into different groups according to their serological properties and macromolecular structure. A comparative analysis of the structure and reactivity of the recombinant monoclonal antibodies showed that the light and heavy Ig-specific chains could form the reactive antibodies when the chains were present in different paratopes of Ig molecules.

[The isolation and characteristics of recombinant monoclonal antibodies to the Lassa virus]. / Poluchenie i kharakteristika rekombinantnykh monoklonal'nykh antitel k virusu Lassa.

Recombinant monoclonal antibodies to Lassa virus were produced. The reactivity of the monoclonal antibodies was studied by indirect fluorescence antibody (IFA) technique, enzyme-linked immunosorbent assay (ELISA), and radioimmunoprecipitation method. The observed reactivity did not correlate with IgG isotyping groups.