Structure-Based Classification Defines the Discrete Conformational Classes Adopted by the Arenaviral GP1.

The emergence of Old and New World arenaviruses from rodent reservoirs persistently threatens human health. The GP1 subunit of the envelope-displayed arenaviral glycoprotein spike complex (GPC) mediates host cell recognition and is an important determinant of cross-species transmission. Previous structural analyses of Old World arenaviral GP1 glycoproteins, alone and in complex with a cognate GP2 subunit, have revealed that GP1 adopts two distinct conformational states distinguished by differences in the orientations of helical regions of the molecule. Here, through comparative study of the GP1 glycoprotein architectures of Old World Loei River virus and New World Whitewater Arroyo virus, we show that these rearrangements are restricted to Old World arenaviruses and are not induced solely by the pH change that is associated with virus endosomal trafficking. Our structure-based phylogenetic analysis of arenaviral GP1s provides a blueprint for understanding the discrete structural classes adopted by these therapeutically important targets.IMPORTANCE The genetically and geographically diverse group of viruses within the family Arenaviridae includes a number of zoonotic pathogens capable of causing fatal hemorrhagic fever. The multisubunit GPC glycoprotein spike complex displayed on the arenavirus envelope is a key determinant of species tropism and a primary target of the host humoral immune response. Here, we show that the receptor-binding GP1 subcomponent of the GPC spike from Old World but not New World arenaviruses adopts a distinct, pH-independent conformation in the absence of the cognate GP2. Our analysis provides a structure-based approach to understanding the discrete conformational classes sampled by these therapeutically important targets, informing strategies to develop arenaviral glycoprotein immunogens that resemble GPC as presented on the mature virion surface.

Comparative analysis of disease pathogenesis and molecular mechanisms of New World and Old World arenavirus infections.

Arenaviruses can cause fatal human haemorrhagic fever (HF) diseases for which vaccines and therapies are extremely limited. Both the New World (NW) and Old World (OW) groups of arenaviruses contain HF-causing pathogens. Although these two groups share many similarities, important differences with regard to pathogenicity and molecular mechanisms of virus infection exist. These closely related pathogens share many characteristics, including genome structure, viral assembly, natural host selection and the ability to interfere with innate immune signalling. However, members of the NW and OW viruses appear to use different receptors for cellular entry, as well as different mechanisms of virus internalization. General differences in disease signs and symptoms and pathological lesions in patients infected with either NW or OW arenaviruses are also noted and discussed herein. Whilst both the OW Lassa virus (LASV) and the NW Junin virus (JUNV) can cause disruption of the vascular endothelium, which is an important pathological feature of HF, the immune responses to these related pathogens seem to be quite distinct. Whereas LASV infection results in an overall generalized immune suppression, patients infected with JUNV seem to develop a cytokine storm. Additionally, the type of immune response required for recovery and clearance of the virus is different between NW and OW infections. These differences may be important to allow the viruses to evade host immune detection. Understanding these differences will aid the development of new vaccines and treatment strategies against deadly HF viral infections.

Differential recognition of Old World and New World arenavirus envelope glycoproteins by subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P).

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residue Y285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

Ocozocoautla de espinosa virus and hemorrhagic fever, Mexico.

Arenavirus RNA was isolated from Mexican deer mice (Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.

Novel arenavirus infection in humans, United States.

Immunoglobulin G against Whitewater Arroyo virus or lymphocytic choriomeningitis virus was found in 41 (3.5%) of 1,185 persons in the United States who had acute central nervous system disease or undifferentiated febrile illnesses. The results of analyses of antibody titers in paired serum samples suggest that a North American Tacaribe serocomplex virus was the causative agent of the illnesses in 2 persons and that lymphocytic choriomeningitis virus was the causative agent of the illnesses in 3 other antibody-positive persons in this study. The results of this study suggest that Tacaribe serocomplex viruses native to North America, as well as lymphocytic choriomeningitis virus, are causative agents of human disease in the United States.

Investigation of clade B New World arenavirus tropism by using chimeric GP1 proteins.

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.

Structure of Machupo virus polymerase in complex with matrix protein Z.

The Arenaviridae family includes several viruses that cause severe human hemorrhagic fevers with high mortality, with no effective countermeasures currently available. The arenavirus multi-domain L protein is involved in viral transcription and replication and represents a promising target for antiviral drugs. The arenavirus matrix protein Z is a small multi-functional protein that inhibits the activities of the L protein. Here we report the structure of Machupo virus L protein in complex with Z determined by cryo-electron microscopy. The Z protein acts as a staple and binds the L protein with 1:1 stoichiometry at the intersection between the PA-C-like region, RNA-dependent RNA polymerase and PB2-N-like region. Binding of the Z protein may lock the multiple domains of L into a fixed arrangement leading to loss of catalytic activity. These results further our understanding of the inhibitory mechanism of arenavirus replication machinery and provide a novel perspective to develop antiviral drugs.

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

Genetic diversity among Bolivian arenaviruses.

Machupo virus and Chapare virus are members of the Tacaribe serocomplex (virus family Arenaviridae) and etiological agents of hemorrhagic fever in humans in Bolivia. The nucleotide sequences of the complete Z genes, a large fragment of the RNA-dependent RNA polymerase genes, the complete glycoprotein precursor genes, and the complete nucleocapsid protein genes of 8 strains of Machupo virus were determined to increase our knowledge of the genetic diversity among the Bolivian arenaviruses. The results of analyses of the predicted amino acid sequences of the glycoproteins of the Machupo virus strains and Chapare virus strain 200001071 indicated that immune plasma from hemorrhagic fever cases caused by Machupo virus may prove beneficial in the treatment of Bolivian hemorrhagic fever but not hemorrhagic fever caused by Chapare virus.

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana).

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.

Catarina virus, an arenaviral species principally associated with Neotoma micropus (southern plains woodrat) in Texas.

The purpose of this study was to define the taxonomic relationship of an arenavirus principally associated with the southern plains woodrat (Neotoma micropus) in southern Texas to other New World arenaviruses. The results of independent analyses of glycoprotein precursor amino acid sequences and nucleocapsid protein amino acid sequences indicated that the arenavirus in southern Texas is novel (proposed species name Catarina virus) and phylogenetically most closely related to Whitewater Arroyo virus, which is principally associated with the white-throated woodrat (Neotoma albigula) in northwestern New Mexico. Together, the close phylogenetic relationship between Catarina virus and Whitewater Arroyo virus and the association of these viral species with congeneric rodent species support the notion that the principal host relationships of some New World arenaviruses are a product of a long-term shared evolutionary relationship between the virus family Arenaviridae and the rodent family Cricetidae.

Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses.

BACKGROUND: Five of the known arenaviruses cause viral hemorrhagic fever in humans and are classified as biosafety level 4 pathogens. Four of the viruses, namely Junin, Guanarito, Machupo, and Sabia, belong to clade B of New World arenaviruses that also comprises the nonpathogenic viruses Tacaribe, Cupixi, and Amapari. OBJECTIVES: To establish real-time reverse transcription (RT)-PCR assays for Junin and Guanarito virus based on fluorescence resonance energy transfer (FRET) probes, and a universal RT-PCR assay for all known clade B viruses with conventional read-out. RESULTS: Conserved sequences in the nucleoprotein gene were chosen as target sites for primers and FRET probes. A common set of primers was designed for all three assays. The assays were based on one-step RT-PCR reagents and were optimised with respect to analytical sensitivity using synthetic RNA templates. The real-time PCR assays detected about 0.5 and 5TCID(50) of cell culture-derived Junin and Guanarito virus, respectively. The universal clade B PCR amplified cell culture-derived RNA of Junin, Guanarito, Machupo, and Sabia virus (5-500TCID(50) per reaction), as well as RNA of Tacaribe, Cupixi, and Amapari virus. CONCLUSIONS: The PCR assays may be used as complementary diagnostic tests for pathogenic New World arenaviruses. The universal PCR assay could also be suitable for the detection of novel clade B arenaviruses in patients as well as in animal reservoirs.

Co-circulation of Clade C New World Arenaviruses: New geographic distribution and host species.

Clade C, of the New World Arenaviruses, is composed of only the Latino and Oliveros viruses and, besides the geographic range of their rodent reservoirs, the distribution of these viruses has been restricted to Bolivia and Argentina. In this study, the genetic detection and phylogenetic analysis of the complete S segment sequences of sympatric arenaviruses from Brazil revealed a new geographic distribution of clade C arenaviruses, as well as the association of Oliveros virus with a new rodent reservoir.

Pinhal Virus, a New Arenavirus Isolated from Calomys tener in Brazil.

Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

Phylogeny of the Venezuelan 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 Pichindé virus (a South American arenavirus which, like Pirital virus, does not appear to be pathogenic for humans) and placed the Pirital-Pichindé 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.

Natural nidality in Bolivian hemorrhagic fever and the systematics of the reservoir species.

Zoonoses within wild reservoir host populations often occur focally obeying Pavlovskii's rules of "natural nidality". What appears to be a clear example is Bolivian hemorrhagic fever (BHF), a disease endemic to northeastern Bolivia. The etiological agent is Machupo virus (MACV, Arenaviridae). The vertebrate reservoir, identified 30 years ago, was Calomys callosus a wild rodent common to open biomes in the lowlands of southeastern South America. The lack of concordance between the occurrence of MACV and the range of its rodent host has puzzled cadres of researchers and could be used as an exemplar of natural nidality. Here, we show that the populations of rodents responsible for the maintenance and transmission of MACV are an independent monophyletic lineage, different from those in other areas of South America. Therefore a clearer understanding of the systematics of the host species explains the apparent natural nidality of BHF. Similar studies may prove to be informative in other zoonoses.

Arenaviruses other than Lassa virus.

The family Arenaviridae includes 23 viral species, of which 5 can cause viral hemorrhagic fevers with a case fatality rate of about 20%. These five viruses are Junin, Machupo, Guanarito, Sabia and Lassa virus, the manipulation of which requires biosafety level 4 facilities. They are included in the Category A Pathogen List established by the Center for Disease Control and Prevention that groups agents with the greatest potential for adverse public health impact and mass casualties whether a situation characterized by a ill-intentioned abuse of natural or engineered arenavirus would be encountered. The aims of this article are to (i) summarize the current situation; (ii) provide information to help anticipating the effects to be expected in such a situation; and to (iii) emphasize the need for fundamental research to allow the development of diagnostic, prevention and therapeutic tools as countermeasures to weaponized arenaviruses.

Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus).

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

Epizootiology of arenaviral infections in the white-throated woodrat (Muridae: Sigmodontinae) and other woodrats in Arizona.

The purpose of this study was to extend and refine our knowledge of the geographical distribution and natural host relationships of the arenaviruses associated with woodrats indigenous to Arizona. Antibody to a Tacaribe serocomplex virus was found in 112 of 1,250 white-throated woodrats, five of 208 Mexican woodrats, one of 114 Stephen's woodrats, and none of 862 other rodents captured at 51 sites in 10 counties in Arizona. Of the 112 antibody-positive white-throated woodrats, 109 (97.3%) were captured within extensive, dense patches of prickly pear cactus and cane cholla in three counties in mid-central Arizona. Analysis of the serological and zoographical data suggested that white-throated woodrats usually become infected early in life and that the distribution of antibody-positive white-throated woodrats in Arizona is not linked to a specific biome.