The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.

Electrochemical determination of hantavirus using gold nanoparticle-modified graphene as an electrode material and Cu-based metal-organic framework assisted signal generation.

An electrochemical biosensor was prepared for nucleic acid-based hantavirus detection using a Cu-based metal-organic framework (CuMOF) as a signal tag. The CuMOF was synthesized by the solvothermal method and then covalently bonded with signal DNA (sDNA) probes. The Au nanoparticles and reduced graphene oxide composite were deposited on the electrode surface by electroreduction as support substrate and was then functionalized with capture DNA (cDNA) probes by self-assembly. Through the complementary base pairing, the target DNA (tDNA) fragment of hantavirus hybridized with the cDNA and the sDNA in a sandwich-type format. The tDNA was detected according to the current signal of the CuMOF catalyzed reaction using o-phenylenediamine as redox substrate. The peak current of the biosensor at - 0.55 V increased linearly in proportion to the logarithmic value of the tDNA concentration from 10-15 to 10-9 mol/L, with a detection limit of 0.74 × 10-15 mol/L. Moreover, the proposed biosensor was successfully applied to detect hantavirus and was able to distinguish hantavirus from other arboviruses.

The Postfusion Structure of the Heartland Virus Gc Glycoprotein Supports Taxonomic Separation of the Bunyaviral Families Phenuiviridae and Hantaviridae.

Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low-pH conditions. Here, we describe the crystal structure of HRTV Gc in its postfusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct features in the aspects of subdomain orientation, N-linked glycosylation, the interaction pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens.IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee on Taxonomy of Viruses reclassification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases.

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5' end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

UNLABELLED: Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE: Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection.

Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

Interactions and oligomerization of hantavirus glycoproteins.

In this report the basis for the structural architecture of the envelope of hantaviruses, family Bunyaviridae, is systematically studied by the interactions of two glycoproteins N and C (Gn and Gc, respectively) and their respective disulfide bridge-mediated homo- and heteromeric oligomerizations. In virion extracts Gn and Gc associated in both homo- and hetero-oligomers which were, at least partially, thiol bridge mediated. Due to strong homo-oligomerization, the hetero-oligomers of Gn and Gc are likely to be mediated by homo-oligomeric subunits. A reversible pH-induced disappearance of a neutralizing epitope in Gc and dissociation of the Gn-Gc complex at pH values below 6.2 provide proteochemical evidence for the fusogenicity of Gc. Incomplete inactivation of virions at acidic pH indicates that additional factors are required for hantavirus fusion, as in the case of pestiviruses of the Flaviviridae. Based on similarities to class II fusion proteins, a structure model was created of hantavirus Gc using the Semliki Forest virus E1 protein as a template. In total, 10 binding regions for Gn were found by peptide scanning, of which five represent homotypic (Gn(I) to Gn(V)) and five represent heterotypic (Gc(I) to Gc(V)) interaction sites that we assign as intra- and interspike connections, respectively. In conclusion, the glycoprotein associations were compiled to a model wherein the surface of hantaviruses is formed of homotetrameric Gn complexes interconnected with Gc homodimers. This organization would create the grid-like surface pattern described earlier for hantaviruses in negatively stained electron microscopy specimens.

Hantavirus Replication Cycle-An Updated Structural Virology Perspective.

Hantaviruses infect a wide range of hosts including insectivores and rodents and can also cause zoonotic infections in humans, which can lead to severe disease with possible fatal outcomes. Hantavirus outbreaks are usually linked to the population dynamics of the host animals and their habitats being in close proximity to humans, which is becoming increasingly important in a globalized world. Currently there is neither an approved vaccine nor a specific and effective antiviral treatment available for use in humans. Hantaviruses belong to the order Bunyavirales with a tri-segmented negative-sense RNA genome. They encode only five viral proteins and replicate and transcribe their genome in the cytoplasm of infected cells. However, many details of the viral amplification cycle are still unknown. In recent years, structural biology methods such as cryo-electron tomography, cryo-electron microscopy, and crystallography have contributed essentially to our understanding of virus entry by membrane fusion as well as genome encapsidation by the nucleoprotein. In this review, we provide an update on the hantavirus replication cycle with a special focus on structural virology aspects.

The Serological Cross-Detection of Bat-Borne Hantaviruses: A Valid Strategy or Taking Chances?

Bats are hosts of a range of viruses, and their great diversity and unique characteristics that distinguish them from all other mammals have been related to the maintenance, evolution, and dissemination of these pathogens. Recently, very divergent hantaviruses have been discovered in distinct species of bats worldwide, but their association with human disease remains unclear. Considering the low success rates of detecting hantavirus RNA in bat tissues and that to date no hantaviruses have been isolated from bat samples, immunodiagnostic tools could be very helpful to understand pathogenesis, epidemiology, and geographic range of bat-borne hantaviruses. In this sense, we aimed to identify in silico immunogenic B-cell epitopes present on bat-borne hantaviruses nucleoprotein (NP) and verify if they are conserved among them and other selected members of Mammantavirinae, using a combination of (the three most used) different prediction algorithms, ELLIPRO, Discotope 2.0, and PEPITO server. To support our data, we in silico modeled 3D structures of NPs from representative members of bat-borne hantaviruses, using comparative and ab initio methods due to the absence of crystallographic structures of studied proteins or similar models in the Protein Data Bank. Our analysis demonstrated the antigenic complexity of the bat-borne hantaviruses group, showing a low sequence conservation of epitopes among members of its own group and a minor conservation degree in comparison to Orthohantavirus, with a recognized importance to public health. Our data suggest that the use of recombinant rodent-borne hantavirus NPs to cross-detect antibodies against bat- or shrew-borne viruses could underestimate the real impact of this virus in nature.

Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein.

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The interaction was initially established with native proteins by co-immunoprecipitating PUUV nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to bind to the predicted CT of Gc. The importance of Gn- and Gc-CTs to the recognition of RNP was further verified in pull-down assays using soluble peptides with binding capacity to both recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species, suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our previous results, we suggest that the complete hetero-oligomeric (Gn-Gc)(4) spike complex of hantaviruses mediates the packaging of RNP into virions.

Diverse Morphology and Structural Features of Old and New World Hantaviruses.

To further understanding of the structure and morphology of the Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus.

Quick electrochemical immunoassay for hantavirus detection based on biochar platform.

This work describes the first method using biochar (BC) as carbonaceous platform for immunoassay application. BC is a highly functionalized material obtained through biomass pyrolysis under controlled conditions. Due to the highly functionalized surface, covalent binding between BC and biomolecules can be performed by EDC/NHS conjugation. The application of the modified electrode was done with Hantavirus, that are etiologic agents mainly transmitted by wild rodents. Among its pathologies Hantavirus Cardiopulmonary Syndrome (HCPS) arises at Americas, caused by Hantavirus Araucária and reaches 40% lethality. The diagnostic is based on the presence of specific hantavirus nucleoprotein (Np), under viremic condition or IgG2b antibodies (Ab), during first symptoms. The results presented a device sensitivity of 5.28 µA dec-1 and a LOD of 0.14 ng mL-1 to the Np detection, ranging from 5.0 ng mL-1 to 1.0 µg mL-1, the Ab detection works as qualitative type sensor above 200 ng mL-1. Both sensors were evaluated its selectivity and serum samples; selectivity against Gumboro disease, VP2 protein, and antibody IgG2a against Yellow fever disease (YF), respectively. So, the devices here proposed are promising tool suitable for both rodent and human hantavirus clinical surveys.

Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses.

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

BACKGROUND: The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. RESULTS: In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). CONCLUSION: Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

Recombinant DNA vaccine of Hantavirus Gn and LAMP1 induced long-term immune protection in mice.

BACKGROUND: Prophylaxis is widely adopted the best choice against Hemorrhagic fever with renal syndrome (HFRS) caused by Hantavirus. However, loss of memory immune response maintenance remains as major shortcoming in current HFRS vaccine. A recombinant DNA vaccine, pVAX-LAMP/Gn was previously proved efficient, requiring long-term evaluations. METHODS & RESULTS: Immune responses of Balb/c mice were assessed by specific and neutralizing antibodies, interferon-γ ELISpot assay, and cytotoxic T-lymphocyte cytotoxicity assay. HTNV-challenge assay identified long-term protection. Safety was confirmed by histological and behavioral analysis. Epitope-spreading phenomenon was noted, revealing two sets of dominant T-cell epitopes cross-species. CONCLUSION: pVAX-LAMP/Gn established memory responses within a long-term protection. Lysosome-targeted strategy showed promise on Gn-based DNA vaccine and further investigations are warranted in other immunogenic Hantaviral antigens.

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

Comparison of the deduced gene products of the L, M and S genome segments of hantaviruses.

The amino acid sequences deduced from all currently available nucleotide sequences of hantaviruses are compared. Comparisons of three large (L), eight medium (M) and five small (S) genome segments are included. A consensus sequence is provided, allowing easy identification of conserved and unique gene regions. The viruses included in this report represent four serologically distinct hantaviruses which are capable of causing severe, moderate, mild or no human disease.

Genetic characterization and phylogeny of Andes virus and variants from Argentina and Chile.

Andes virus, one of five hantaviruses known to cause hantavirus pulmonary syndrome (HPS), emerged in 1995 in southwestern Argentina (López et al. (1996) Virology 220, 223-226). The complete nucleotide sequence of Andes virus S genome segment was determined and compared with sequences of viral RNAs in autopsy tissues of more recently reported HPS cases from southwestern Argentina and south of Chile (cases ESQ H-1/96 and CH H-1/96). Andes virus S segment was found to be 1876 nucleotides in length and to encode the nucleocapsid protein (N), 428 amino acids in length. S segment analysis also revealed a long 5' non-coding region (547 nucleotides) which displays three copies of an octanucleotide sequence repeat. Comparisons of S segment sequences of ESQ H-1/96 and CH H-1/96 (82% of the entire genome sequence) with the corresponding sequences of Andes virus revealed identities of 97.2% and 98.5%, respectively. Sequence motifs identical and in the same positions as exhibited in Andes virus 5' non-coding region were found in both, ESQ H-1/96 and CH H-1/96 sequences. Three genome fragments of the M segment sequence of the viruses (representing approximately 34% of the entire sequence) were also analyzed. Comparisons of S and M segment sequences of Andes virus with the corresponding sequences of ESQ H-1/96 showed S and M segment identities which differ by less than 1.4%. Andes virus and CH H-1/96 have S segments that differ by 1.5% from one another while their M segment fragments differ by 5.5-8.2%. Phylogenetic analysis showed that Andes virus along with ESQ H-1/96 and CH H-1/96 form a distinct lineage within the clade containing Bayou and Black Creek Canal viruses. It also showed that Andes virus branch of trees derived from comparisons of S or M sequences differed. It is concluded that Andes virus variants causing HPS circulate east and west of the Andes mountains.