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The southern plains woodrat (Neotoma micropus) is the principal host of Catarina virus in southern Texas and a natural host of other North American Tacaribe serocomplex viruses. The objectives of this study were to increase our knowledge of the genetic diversity among Tacaribe serocomplex viruses associated with N. micropus and to define better the natural host relationships of these viruses. Pairwise comparisons of complete glycoprotein precursor gene sequences and complete nucleocapsid protein gene sequences revealed a high level of genetic diversity among Tacaribe serocomplex viruses associated with N. micropus in western Oklahoma, southern New Mexico, and northern and southern Texas. Collectively, the results of Bayesian analyses of nucleotide sequences and pairwise comparisons of amino acid sequences confirmed that the arenaviruses associated with N. micropus in Oklahoma and New Mexico should be included in the Whitewater Arroyo species complex, and indicated that that the arenaviruses associated with N. micropus in northern Texas are strains of a novel arenaviral species – tentatively named “Middle Pease River virus”. Together, the results of assays for arenavirus and assays for anti-arenavirus antibody in 54 southern plains woodrats and 325 other rodents captured at 2 localities suggested that the southern plains woodrat is the principal host of Middle Pease River virus in northern Texas.

In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.

In the present paper the in vitro antiviral activity of dehydroepiandrosterone (DHEA), epiandrosterone (EA) and 16 synthetic derivatives against Junin virus (JUNV) replication in Vero cells was studied. DHEA and EA caused a selective inhibition of the replication of JUNV and other members of the Arenaviridae family such as Pichinde virus and Tacaribe virus. The compounds were not virucidal to cell-free JUNV. The impairment of viral replication was not due to an inhibitory effect of the steroids on virus adsorption or internalization. An inhibitory effect of the compounds on JUNV protein synthesis and both intracellular and extracellular virus production was demonstrated. A partial inhibitory action on cell surface expression of JUNV glycoprotein G1 was also detected on DHEA- and EA-treated cultures. Like DHEA and EA, three compounds obtained from EA by chemical synthesis showed selectivity indexes higher than ribavirin, the only antiviral compound that has shown partial efficacy against arenavirus infections.

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus.

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

The influence of glycoprotein processing, cleavage and transport on Junin virus (JV) infectivity was investigated using monensin combined with lectin binding assays. Yields of extracellular virus were more significantly reduced than cell-associated virus, indicating that monensin inhibited the transport of infectious virus to the extracellular space on a late stage of the replicative cycle. Shown by lectin reactivity and immunoprecipitation, the intracellular processing of JV glycoproteins involved first the maturation of GPC oligosaccharides to a complex form and then the precursor cleavage which might occur late in transit through or exit from the Golgi cisternae. Cleavage of GPC to yield the mature GP38 as well as cell surface immunofluorescence were blocked by monensin. Thus, GP38 production together with glycoprotein transport to the cell membrane seemed to be required for the release of infectious virus from JV-infected cells.

Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junin virus, one of the few human pathogenic arenaviruses. The S RNA of Junin virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junin virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junin virus RNA sequences present in a single infected BHK21 cell over a background of 10 4 uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junin virus in RNA extracted from 100 μl of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junin virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.

The early events in Junin virus (JUNV) infection are not thoroughly understood. We have previously shown that JUNV enter cells by clathrin-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early virus infection. Inhibitory effects of drugs affecting main cytoskeletal components on JUNV entry into Vero cells were analyzed. Drugs that disrupted microfilaments or stabilized microtubules inhibited early steps of virus entry. In contrast, drugs that stabilized microfilaments or depolymerized microtubules were not able to block virus entry very efficiently. Furthermore, real time PCR was performed to detect viral entry and we found more than 10-fold less RNA when microfilaments were depolymerized while a 100-fold diminution was seen when microtubules were stabilized. Taken together our results demonstrate that JUNV relies on an intact actin network during early infection in Vero cells while a dynamic microtubule network is also needed. This represents an important contribution to the characterization of arenavirus multiplication cycle.

The genome of Tacaribe virus (TV), prototype of the New World arenaviruses, comprises two RNA segments each encoding two proteins in an ambisense orientation separated by an intergenic region (IGR). We used a TV minireplicon system to investigate the nature of the IGR structures required for transcription termination. We show that efficient generation of subgenomic (SG) RNAs is related to a single hairpin structure comprising a stem with variable numbers of uninterrupted base pairs and stabilized by high Δ G values. The low ability of highly stable hairpin structures comprising bulged stems to support SG RNA synthesis suggested the importance of hairpin configuration for transcription termination. Neither the sequences downstream nor those upstream from the hairpin played a role in SG RNA accumulation. We also show that independently of the IGR structure the unencapsidated mRNAs contained short stretches of nontemplated bases at their 5′ ends which are capped, whereas the 5′ ends of the nucleocapsid-associated antiminigenomes contained an uncapped extra residue. The results support the conclusions that: (i) transcription termination in TV is related to a structural element that is independent of sequence and (ii) the transcription termination signal is not required for a correct initiation of transcription and replication.

Viral hemorrhagic fevers represent serious human public health problems causing devastating and often lethal disease. Several hemorrhagic fevers are caused by arenaviruses including Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In recent years, increased air travel between Africa and other areas has led to the importation of LFV into the US, Europe, Japan, and Canada. This has raised awareness about arenaviruses as potential emerging viruses. Moreover, because of its severe morbidity and high mortality, and transmissibility from human to human, weaponized forms of LFV poses a real threat as agent of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these infections. Therefore, the importance of developing novel effective antiviral drugs to combat HF arenaviruses, for which the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) provides us with an excellent model system. Recent findings have shown that LCMV multiplication both in cultured cells and in vivo is highly susceptible to the mutagenic agent 5-fluorouracil (FU). FU-mediated extinction of LCMV was associated with only modest increases in virus mutation frequencies, but did not significantly affect virus replication and transcription, or virus particle formation. These findings indicate that, as with other riboviruses, lethal mutagenesis is effective also against LCMV raising the possibility of using this novel antiviral strategy to combat pathogenic arenaviruses.

Guanarito virus (the etiologic agent of Venezuelan hemorrhagic fever, VHF) and Pirital virus coexist in the region of Venezuela in which Venezuelan hemorrhagic fever is endemic. The purpose of this study was to extend our knowledge of the evolutionary relationship between these two arenaviruses. We determined that the large genomic segments of Guanarito virus and Pirital virus are similar in size and identical in structural organization to the large genomic segments of other South American arenaviruses. For example, the Z proteins and RNA-dependent RNA polymerases of Guanarito virus and Pirital virus are encoded in nonoverlapping open reading frames of opposite polarities. Phylogenetic analyses of Z protein gene nucleotide sequences and RNA-dependent RNA polymerase gene nucleotide sequences grouped Pirital virus with Pichinde virus (a South American arenavirus which, like Pirital virus, does not appear to be pathogenic for humans) and placed the Pirital-Pichinde lineage in a sister relationship to a lineage represented by Guanarito virus and the three other arenaviruses known to cause hemorrhagic fever in South America. These results are concordant with the results of studies on the phylogeny of the arenavirus small genomic segment. Thus, the exchange of genomic elements between Guanarito virus and Pirital virus via recombination or reassortment likely did not contribute to the emergence of Venezuelan hemorrhagic fever.

RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junin virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

The completed sequence of the arenavirus, lymphocytic choriomeningitis virus, revealed a new gene encoding a small protein with a single zinc-binding domain. The cDNA for this gene has been expressed in E. coli to produce fusion protein that has been used to raise antisera. The antisera facilitated the positive identification of the p11 ‘Z’ gene product as a structural component of the virion. A related arenavirus, Tacaribe, has a comparable p11 gene product. The abundance of the p11 Z protein relative to other virion components has been determined by metabolic labeling. Triton X-114 extraction and dimethyl suberimidate-HCl crosslinking indicate that the p11 Z protein is a hydrophobic protein associated with the nucleocapsid of the virion core.

The nucleotide sequence of the small (S) genomic RNA of Lassa virus (strain GA391, of Nigerian origin) has been determined. The RNA has features which conform to those seen in most other arenavirus S RNAs which have been characterised, including conserved terminal sequences, an ambisense arrangement of the coding regions for the precursor glycoprotein (GPC) and nucleocapsid (N) proteins and an intergenic region capable of forming a base-paired "hairpin" structure. Comparison of the nucleotide sequence with that of the Josiah strain of Lassa virus (from Sierra Leone) reveals considerable nucleotide divergence in the third base of codons in the reading frames of all three proteins, although the resulting protein sequences are highly conserved, with 92, 94 and 91% identical residues for the mature glycoproteins G1 and G2 and the N protein, respectively. Sequence alignments of the available arenavirus structural proteins and dendrograms summarising the relationships between the viral proteins are presented.

Previous studies have reported that the 3' half of the small, S, RNA species of the WE strain of lymphocytic choriomeningitis (LCM) virus codes for the viral nucleoprotein in a subgenomic, viral-complementary, mRNA species (Romanowski, V. and Bishop, D.H.L. (1985) Virus Res. 2, 35-51). The complete sequence of the LCM-WE S RNA has now been obtained, indicating that the 5' half of the RNA codes for the viral glycoprotein precursor in a viral-sense sequence that does not overlap the N gene. It is concluded that, like Pichinde virus (Auperin, D. et al. (1984) J. Virol. 52, 897-904), LCM has an ambisense S RNA coding strategy. The LCM-WE S RNA is 3375 nucleotides in length, has a size of 1.14 X 10(6) Da and base composition of 26.1% A, 23.2% C, 21.5% G, 29.2% U. The 3' and 5' end sequences of the S RNA are complementary for some 30 nucleotides, depending on the arrangement. The non-coding regions at the two ends are 77 (5') and 60 (3') nucleotides long. The glycoprotein precursor has a primary amino acid size of 56293 Da and is rich in potential glycosylation sites as well as histidine and cysteine residues. It has both amino and carboxy proximal hydrophobic regions. The LCM-WE S RNA and predicted protein sequence data have been compared to those of Pichinde arena-virus. Extensive RNA and protein sequence homology exists for the two S RNA species, although the homology for the glycoprotein sequences of the two viruses (39%) is less than the 50% observed for the two viral nucleoproteins.

In an attempt to define the involvement of host transcription in arenavirus growth, a study was made of the effect of actinomycin D (AMD) on the yields of infectious Pichinde, Tacaribe and Junin viruses. The drug was added either immediately after virus adsorption or later after infection, at the stationary phase of virus growth. The time of exposure of the infected cells to the inhibitor was so chosen that the generation and release of virus into the medium took place in the presence of AMD. A double label technique was used to estimate the generation time of an arenavirus particle. This was found to be 6 h or less for all of the arenaviruses examined. The results indicated that treatment of the host cells with AMD, either immediately after virus adsorption or later after infection, does not affect the yield of infectious Pichinde, Tacaribe or Junin viruses, thus implying that continuous host transcription is not required for the replication cycle of these arenaviruses.

Analyses of the 3' end sequences of the small, S, and large, L, RNA species of lymphocytic choriomeningitis (LCM) virus isolates ARM and WE, and DNA clones of LCM-WE, have shown that there are extensive RNA sequence homologies between the 3' ends of the two RNA species of both LCM strains. Limited sequence data of DNA clones representing the LCM-WE L RNA species indicate that a gene product (presumably the minor 200 kdalton virion protein) is coded in a viral-complementary mRNA species. Sequence analyses of LCM-WE S DNA clones indicate that approximately 50% of the 2040 nucleotides representing the 3' half of the viral RNA species (and its encoded 558 amino acid gene product) are identical in type and position to those of Pichinde arenavirus (Auperin, D., et al. (1984a), Virology 134, 208-219). For Pichinde virus, it has been shown that the 3' proximal gene product (the nucleoprotein, N) is translated from a subgenomic, viral-complementary mRNA (Auperin et al., 1984a). Data have recently been obtained (Auperin, D., et al. (1984b) J. Virol., in press) that indicate that the Pichinde glycoprotein precursor, GPC, is coded in a viral-sense subgenomic mRNA species corresponding to the 5' half of the S RNA. The nucleotide sequence that immediately follows the N coding region of both LCM-WE and Pichinde viruses can be arranged in a hairpin configuration. In view of this, and if, like Pichinde virus, LCM has an ambisense S RNA coding strategy, then it is probable that the intergenic hairpins function as transcription terminators for the N and GPC mRNA species of both viruses.

C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10 2 PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.