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.

ICTV Virus Taxonomy Profile: Hantaviridae 2024.

Hantaviridae is a family for negative-sense RNA viruses with genomes of about 10.5-14.6 kb. These viruses are maintained in and/or transmitted by fish, reptiles, and mammals. Several orthohantaviruses can infect humans, causing mild, severe, and sometimes-fatal diseases. Hantavirids produce enveloped virions containing three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hantaviridae, which is available at ictv.global/report/hantaviridae.

Protocadherin-1 is essential for cell entry by New World hantaviruses.

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-ß, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Mother-to-Child Transmission of Andes Virus through Breast Milk, Chile1.

Andes virus (ANDV) is the only hantavirus transmitted between humans through close contact. We detected the genome and proteins of ANDV in breast milk cells from an infected mother in Chile who transmitted the virus to her child, suggesting gastrointestinal infection through breast milk as a route of ANDV person-to-person transmission.

[Cell biology of phlebovirus entry]. / Entrée cellulaire des phlébovirus chez l'hôte mammifère.

Phleboviruses constitute a large group of arthropod-borne viruses (arboviruses), mainly transmitted to their hosts by sandflies and ticks, occasionally by mosquitoes. These viruses have a worldwide distribution and many cause serious diseases - often fatal - in both domestic animals and humans. The global warming, the apparent wide distribution of arthropod reservoirs, and the increasing number of outbreaks show that phleboviruses must be taken seriously as emerging disease agents. This review proposes to focus on the early steps of phlebovirus infection, from virus binding to penetration into the cytosol. We address the most recent knowledge and advances in the entry of these viruses into vertebrate host cells, including virus receptors, cellular factors, endocytic pathways, and fusion.

Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation.

Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.

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.

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.

Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

Hantavirus-infection confers resistance to cytotoxic lymphocyte-mediated apoptosis.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

Pending Reorganization of Hantaviridae to Include Only Completely Sequenced Viruses: A Call to Action.

The official classification of newly discovered or long-known unassigned viruses by the International Committee on Taxonomy of Viruses (ICTV) requires the deposition of coding-complete or -near-complete virus genome sequences in GenBank to fulfill a requirement of the taxonomic proposal (TaxoProp) process. However, this requirement is fairly new; thus, genomic sequence information is fragmented or absent for many already-classified viruses. As a result, taxon-wide modern phylogenetic analyses are often challenging, if not impossible. This problem is particularly eminent among viruses with segmented genomes, such as bunyavirals, which were frequently classified solely based on single-segment sequence information. To solve this issue for one bunyaviral family, Hantaviridae, we call on the community to provide additional sequence information for incompletely sequenced classified viruses by mid-June 2023. Such sequence information may be sufficient to prevent their possible declassification during the ongoing efforts to establish a coherent, consistent, and evolution-based hantavirid taxonomy.

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host.

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.

Induction of SARS-CoV-2 neutralizing antibodies by CoronaVac and BNT162b2 vaccines in naïve and previously infected individuals.

BACKGROUND: A major challenge of the SARS-CoV-2 pandemic is to better define "protective thresholds" to guide the global response. We aimed to characterize the longitudinal dynamics of the antibody responses in naturally infected individuals in Chile and compared them to humoral responses induced after immunization with CoronaVac-based on an inactivated whole virus -or the BNT162b2- based on mRNA-vaccines. We also contrasted them with the respective effectiveness and efficacy data available for both vaccines. METHODS: We determined and compared the longitudinal neutralizing (nAb) and anti-nucleocapsid (anti-N) antibody responses of 74 COVID-19 individuals (37 outpatient and 37 hospitalized) during the acute disease and convalescence. We also assessed the antibody boosting of 36 of these individuals who were immunized after convalescence with either the CoronaVac (n = 30) or the BNT162b2 (n = 6) vaccines. Antibody titres were also measured for 50 naïve individuals immunized with two doses of CoronaVac (n = 35) or BNT162b2 (n = 15) vaccines. The neutralizing level after vaccination was compared to those of convalescent individuals and the predicted efficacy was estimated. FINDINGS: SARS-CoV-2 infection induced robust nAb and anti-N antibody responses lasting >9 months, but showing a rapid nAb decay. After convalescence, nAb titres were significantly boosted by vaccination with CoronaVac or BNT162b2. In naïve individuals, the calculated mean titre induced by two doses of CoronaVac or BNT162b2 was 0·2 times and 5.2 times, respectively, that of convalescent individuals, which has been proposed as threshold of protection. CoronaVac induced no or only modest anti-N antibody responses. Using two proposed logistic models, the predicted efficacy of BNT162b2 was estimated at 97%, in close agreement with phase 3 efficacy studies, while for CoronaVac it was ∼50% corresponding to the lowest range of clinical trials and below the real-life data from Chile (from February 2 through May 1, 2021 during the predominant circulation of the Gamma variant), where the estimated vaccine effectiveness to prevent COVID-19 was 62·8-64·6%. INTERPRETATION: The decay of nAbs titres in previously infected individuals over time indicates that vaccination is needed to boost humoral memory responses. Immunization of naïve individuals with two doses of CoronaVac induced nAbs titres that were significantly lower to that of convalescent patients, and similar to vaccination with one dose of BTN162b2. The real life effectiveness for CoronaVac in Chile was higher than estimated; indicating that lower titres and additional cellular immune responses induced by CoronaVac might afford protection in a highly immunized population. Nevertheless, the lower nAb titre induced by two doses of CoronaVac as compared to the BTN162b2 vaccine in naïve individuals, highlights the need of booster immunizations over time to maintain protective levels of antibody, particularly with the emergence of new SARS-CoV-2 variants. FUNDING: FONDECYT 1161971, 1212023, 1181799, FONDECYT Postdoctorado 3190706 and 3190648, ANID Becas/Doctorado Nacional 21212258, PIA ACT 1408, CONICYT REDES180170, Centro Ciencia & Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia grants from the Agencia Nacional de Investigación y Desarrollo (ANID) of Chile; NIH-NIAD grants U19AI135972, R01AI132633 and contracts HHSN272201400008C and 75N93019C00051; the JPB Foundation, the Open Philanthropy Project grant 2020-215611 (5384); and by anonymous donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Aromatic and polar residues spanning the candidate fusion peptide of the Andes virus Gc protein are essential for membrane fusion and infection.

Hantaviruses infect human cells through cell attachment and subsequent fusion of viral and cellular membranes at low pH. This largely unknown entry process is mediated by the Gn and Gc glycoproteins, anchored at the viral envelope membrane. Performing bioinformatic analysis and peptide-liposome-binding assays we suggested in a former report that Gc of Andes virus (ANDV) and other hantaviruses corresponds to the viral fusion protein sharing characteristics with class II fusion proteins. To gain insights into the fusion protein of hantaviruses, residues within the previously predicted fusion peptide of ANDV Gc were substituted and mutant proteins tested in fusion and infection assays. To ensure proper folding of mutant proteins, they were first characterized for trafficking to the plasma membrane and incorporation on to ANDV Gn/Gc-pseudotyped lentiviral particles. Cell attachment of these particles was assessed using a newly developed binding assay and their subsequent entry properties determined by FACS analysis of transduced cells expressing the GFP reporter gene. Furthermore, a three-colour-based cell-cell fusion assay of ANDV Gn/Gc expressing cells was performed. The results indicate an essential role of conserved Gc residues W115 and N118 in membrane fusion. Conversely, substitutions of the non-conserved Gc residue G116 did not considerably affect fusion and infection. Altogether, the findings are fully consistent with our earlier prediction suggesting Gc residues 115-121 as an internal fusion peptide and further emphasize the importance of aromatic and polar residues in hantavirus-cell membrane fusion.

Entry of Phenuiviruses into Mammalian Host Cells.

Phenuiviridae is a large family of arthropod-borne viruses with over 100 species worldwide. Several cause severe diseases in both humans and livestock. Global warming and the apparent geographical expansion of arthropod vectors are good reasons to seriously consider these viruses potential agents of emerging diseases. With an increasing frequency and number of epidemics, some phenuiviruses represent a global threat to public and veterinary health. This review focuses on the early stage of phenuivirus infection in mammalian host cells. We address current knowledge on each step of the cell entry process, from virus binding to penetration into the cytosol. Virus receptors, endocytosis, and fusion mechanisms are discussed in light of the most recent progress on the entry of banda-, phlebo-, and uukuviruses, which together constitute the three prominent genera in the Phenuiviridae family.

Long-lasting neutralizing antibody responses in SARS-CoV-2 seropositive individuals are robustly boosted by immunization with the CoronaVac and BNT162b2 vaccines.

The durability of circulating neutralizing antibody (nAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their boosting by vaccination remains to be defined. We show that outpatient and hospitalized SARS-CoV-2 seropositive individuals mount a robust neutralizing antibody (nAb) response that peaks at days 23 and 27 post-symptom onset, respectively. Although nAb titers remained higher in hospitalized patients, both study groups showed long-lasting nAb responses that can persist for up to 12 months after natural infection. These nAb responses in previously seropositive individuals can be significantly boosted through immunization with two doses of the CoronaVac (Sinovac) or one dose of the BNT162b2 (BioNTech/Pfizer) vaccines, suggesting a substantial induction of B cell memory responses. Noteworthy, three obese previously seropositive individuals failed to mount a booster response upon vaccination, warranting further studies in this population. Immunization of naïve individuals with two doses of the CoronaVac vaccine or one dose of the BNT162b2 vaccine elicited similar levels of nAbs compared to seropositive individuals 4.2 to 13.3 months post-infection with SARS-CoV-2. Thus, this preliminary evidence suggests that both, seropositive and naïve individuals, require two doses of CoronaVac to ensure the induction of robust nAb titers.

Andes virus antigens are shed in urine of patients with acute hantavirus cardiopulmonary syndrome.

Hantavirus cardiopulmonary syndrome (HCPS) is a highly pathogenic emerging disease (40% case fatality rate) caused by New World hantaviruses. Hantavirus infections are transmitted to humans mainly by inhalation of virus-contaminated aerosol particles of rodent excreta and secretions. At present, there are no antiviral drugs or immunotherapeutic agents available for the treatment of hantaviral infection, and the survival rates for infected patients hinge largely on early virus recognition and hospital admission and aggressive pulmonary and hemodynamic support. In this study, we show that Andes virus (ANDV) interacts with human apolipoprotein H (ApoH) and that ApoH-coated magnetic beads or ApoH-coated enzyme-linked immunosorbent assay plates can be used to capture and concentrate the virus from complex biological mixtures, such as serum and urine, allowing it to be detected by both immunological and molecular approaches. In addition, we report that ANDV-antigens and infectious virus are shed in urine of HCPS patients.