Experimental infection of the cane mouse Zygodontomys brevicauda (family Muridae) with guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever.
Chronic infections in specific rodents appear to be crucial to the long-term persistence of arenaviruses in nature. The cane mouse, Zygodontomys brevicauda, is a natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. The purpose of this study was to elucidate the natural history of Guanarito virus infection in Z. brevicauda. Thirty-nine laboratory-reared cane mice each were inoculated subcutaneously with 3.0 log10 plaque-forming units of the Guanarito virus prototype strain INH-95551. No lethality was associated with infection in any animal, regardless of age at inoculation. The 13 newborn, 14 weanling, and 8 of the 12 adult animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. These findings indicate that Guanarito virus infection in Z. brevicauda can be chronic and thus support the concept that this rodent species is the natural reservoir of Guanarito virus. (+info)
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)
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)
Extreme genetic diversity among Pirital virus (Arenaviridae) isolates from western Venezuela.
Pirital-like virus isolates from rodents collected in a variety of habitats within a six-state area of central Venezuela were analyzed genetically by amplifying a portion of the nucleocapsid protein gene using RT-PCR. Comparisons of the sequences from 30 selected Pirital-like virus isolates demonstrated up to 26% divergence in nucleotide sequences and up to 16% divergence in deduced amino acid sequences. Within the Pirital monophyletic group, 14 distinct lineages or genotypes, differing by at least 6% in nucleotide sequences, were identified. Although sample sizes were small for some lineages, many of the different genotypes were sampled in only one region or locality, suggesting allopatric divergence. Complement fixation tests with representatives of the most divergent Pirital virus lineages failed to delineate multiple species or subtypes within the Pirital clade. These results indicate that the previously proposed 12% nucleocapsid protein amino acid sequence divergence cutoff value for delineating arenavirus species is not appropriate for the entire family. When individual clones were examined from PCR amplicons, a mean of 0.17% sequence diversity vs the consensus sequences was detected, suggesting diverse quasispecies populations within infected rodent hosts. Possible explanations for the extreme genetic diversity within and among Pirital virus populations in infected rodents are discussed. (+info)
Generation of "natural killer cell-escape" variants of Pichinde virus during acute and persistent infections.
Pichinde virus (PV) strain AN 3739 was determined to be sensitive to natural killer (NK) cells in vivo by enhanced replication in NK-cell-depleted mice. An NK-sensitive subclone (PV-NKs1) was serially passed in mice whose NK cells had previously been activated by an interferon inducer, and two plaque isolates were shown to be resistant to NK cells but not to interferon. Inoculation of severe-combined-immunodeficient mice with PV-NKs1 led to a persistent infection resulting in an NK-resistant viral population. This is the first demonstration of the isolation of viral "NK-escape" variants, as defined by the ability of the virus to replicate in vivo. (+info)
Posttranslational modification of alpha-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding.
The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is alpha-dystroglycan (alpha-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of alpha-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for alpha-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of alpha-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of alpha-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for alpha-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for alpha-DG. Hence, LARGE-dependent glycosylation of alpha-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans. (+info)
Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures.
In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function. (+info)
Receptor use by pathogenic arenaviruses.
The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, alpha-dystroglycan (alpha-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that alpha-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for alpha-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of alpha-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on alpha-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of alpha-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for alpha-DG in murine ES cells. These findings highlight the importance of molecules other than alpha-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses. (+info)