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.

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.

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.

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.

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.

Diversity among Tacaribe serocomplex viruses (Family Arenaviridae) associated with the southern plains woodrat (Neotoma micropus).

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.

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.

Drug discovery technologies and strategies for Machupo virus and other New World arenaviruses.

INTRODUCTION: Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and Lujo, and the New World Clade B arenaviruses Machupo (MACV), Junín (JUNV), Guanarito (GTOV), Sabiá (SABV), and Chapare (CHPV). All of these viruses are Risk Group 4 biosafety pathogens. MACV causes human disease outbreak with high case-fatality rates. To date, at least 1,200 cases with ≈200 fatalities have been recorded. AREAS COVERED: This review summarizes available systems and technologies for the identification of antivirals against MACV. Furthermore, the article summarizes animal models that have been used for the in vivo evaluation of novel inhibitors. The article highlights present treatments for arenaviral diseases and provides an overview of efficacious small molecules and other therapeutics reported to date. Finally, the article summarizes strategies to identify novel inhibitors for anti-arenaviral therapy. EXPERT OPINION: New high-throughput approaches to quantitate infection rates of arenaviruses, as well as viruses modified to carry reporter genes, will accelerate compound screens and drug discovery efforts. RNAi, gene expression profiling and proteomics studies will identify host targets for therapeutic intervention. New discoveries in the cell entry mechanism of MACV and other arenaviruses as well as extensive structural studies of arenaviral L and NP could facilitate the rational design of antivirals effective against all pathogenic New World arenaviruses.

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.

Diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses (family Arenaviridae).

The purpose of this study was to extend our knowledge of the genetic diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses. Analyses of glycoprotein precursor gene sequence data separated the North American arenaviruses into 7 major phylogenetic groups. The results of analyses of Z gene and nucleocapsid protein gene sequence data were not remarkably different from the glycoprotein precursor gene tree. In contrast, the tree generated from RNA-dependent RNA polymerase gene sequences differed from the glycoprotein precursor gene tree with regard to phylogenetic relationships among the viruses associated with woodrats captured in the western United States, Texas, or northern Mexico. Further analyses of the polymerase gene sequence data set suggested that the difference in topology was a consequence of incongruence among the gene tree data sets or chance rather than genetic reassortment or recombination between arenaviruses.

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.

Transferrin receptor 1 in the zoonosis and pathogenesis of New World hemorrhagic fever arenaviruses.

At least five New World arenaviruses cause severe human hemorrhagic fevers. These viruses are transmitted to humans through contact with their respective South American rodent hosts. Each uses human transferrin receptor 1 (TfR1) as its obligate receptor. Accidental similarities between human TfR1 and TfR1 orthologs of arenaviral host species enable zoonoses, whereas mice and rats are not infectable because they lack these TfR1 determinants of infection. All pathogenic New World arenaviruses bind to a common region of the apical domain of TfR1. The ability of a New World arenavirus to use human TfR1 is absolutely predictive of its ability to cause hemorrhagic fevers in humans. Nonpathogenic arenaviruses, closely related to hemorrhagic fever arenaviruses, cannot utilize human TfR1 but efficiently enter cells through TfR1 orthologs of their native rodent hosts. Mutagenesis studies suggest that minor changes in the entry glycoproteins of these nonpathogenic viruses may allow human transmission. TfR1 is upregulated as a result of iron sequestration during the acute-phase response to infection, and the severity of disease may result from amplification of viral replication during this response.

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.

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.

Genetic diversity between and within the arenavirus species indigenous to western Venezuela.

The results of analyses of Z, RNA-dependent RNA polymerase, glycoprotein precursor, and nucleocapsid protein gene sequence data suggested that Guanarito virus was the most common cause of Venezuelan hemorrhagic fever in a 7-year period in the 1990s and that the evolution of Pirital virus in association with Sigmodon alstoni (Alston's cotton rat) has occurred at a significantly higher rate than the evolution of Guanarito virus in association with Zygodontomys brevicauda (short-tailed cane mouse) on the plains of western Venezuela. The results of analyses of the primary structures of the glycoproteins of the 8 strains of Guanarito virus isolated from humans suggested that these strains would be highly cross-reactive in neutralization assays. Thus, passive antibody therapy may prove beneficial in the treatment of human disease caused by strains of Guanarito virus that are enzootic in the region in which Venezuelan hemorrhagic fever is endemic.

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.

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.