HANTA VIRUS GC FRAGMENTS INHIBITING THE FUSION OF THE VIRUS WITH A CELL.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to protein fragments that have antiviral activity. Specifically, these protein fragments inhibit hantavirus infection in eukaryotic cells. The protein fragments used in this invention correspond to amino acid sequences derived from the hantavirus Gc fusion protein. Furthermore, the invention provides a way to synthesize the aforementioned Gc fusion protein fragments (from now on referred to as Gc fragments).

Hantaviruses, which belong to the Bunyaviridae family, can also be classified among viruses enveloped with a lipid membrane (double layer of phospholipids), which they acquire upon leaving the infected cell. These enveloped viruses have the advantage of being able to infect a cell without altering the infected cell's membrane. This is possible thanks to the membrane fusion process that occurs between an enveloped virus and a host cell, a prerequisite for viral infection. In addition, virus-cell membrane fusion is an essential step in the infection cycle of all enveloped viruses, such as hantaviruses from the Bunyaviridae family. The invention to be described is related to protein fragments derived from the hantavirus Gc fusion protein. These hantavirus Gc fusion protein fragments are efficient at preventing the fusion of a hantavirus membrane and target cell membranes.

The invention provides hantavirus Gc protein fragments, which may come in the form of a pharmaceutical solution and inhibit hantavirus-mediated membrane fusion. The invention also provides a way to synthesize said Gc fragments and the pharmaceutical solutions containing them, as well allowing for prophylactic or therapeutic use against hantavirus infections. In this regard, the invention provides efficient therapeutic agents against hantavirus infections.

BACKGROUND

Hantaviruses are human pathogens that include more than 20 known species present in many parts of the world. In Asian regions, they cause hemorrhagic fever with renal syndrome (HFRS), with clinical patterns and severity varying by agent; it has a mortality rate of 12%. In Europe, HFRS is associated with mild clinical symptoms and a low mortality rate (0.1 %). In America, hantaviruses cause the hantavirus pulmonary syndrome (HPS), along with cardiovascular complications, causing death in more than 30% of cases. HFRS and HPS are associated with thrombocytopenia and alterations in vascular permeability, which can produce hemorrhagic complications and hypertension. In severe HPS cases, these symptoms are accompanied by pulmonary edema, respiratory failure, and cardiac arrest. On the other hand, in severe cases of HFRS, acute kidney failure can occur (reviewed by Jonsson et at, 2010).

Every year, 150,000-200,000 HFRS cases are reported in Asia, affecting mainly China and South Korea. In America, there are 200-500 HPS cases per year, with a higher frequency in Argentina, Brazil, Chile, and the U.S.

Hantaviruses persist in rodents and insectivores, and their phylogenetic group, geographic distribution and epidemiology resemble those of their natural hosts. It is widely believed that hantaviruses have co-evolved with their hosts. The ecology and evolution of hantaviruses in their hosts have become known only recently. Among rodent hantavirus hosts, only four rodent subfamilies of the Muridae family have been identified. These include the Arvicolinae, Murinae, and Sigmodontinae/Neotominae subfamilies. The hantaviruses representing these phylogenetic groups correspond to the Puumala virus {Arvicolinae family), the Hantaan virus (Murinae family), and the Andes virus (Sigmodontinae family). In this context, the Puumala, Hantaan, and Andes hantaviruses correspond to prototypes that represent the three different phylogenetic groups of the Hantavirus genus currently associated with disease in humans. As a result, they also represent its geographic distribution. Beside these three groups, hantaviruses have also been found in shrews, moles (Soricidae and Talpidae families), and more recently in bats (families from the Chiroptera order). In 1993, a highly human pathogenic a species termed Sin Nombre virus was identified in the United States of America, and in 1995 another, phylogenetically closely related virus termed Andes virus (ANDV) was identified in Argentina and Chile. Since then, many closely related hantaviruses have been identified throughout the Americas.

ANTIVIRAL TREATMENT

There are currently no effective antiviral drugs or therapeutic agents available against hantavirus infections. Therefore, the current treatment is only palliative.

Drugs with wide antiviral activity, such as Ribavirin and Lactoferrin, have been studied. Ribavirin is metabolized into a nucleoside analog that interferes with viral RNA replication, while Lactoferrin apparently inhibits virus-cell fusion. In animal models, hantaviruses are sensitive in vitro and in vivo to both antivirals, with Ribavirin generating the best results. Clinical studies showed that Ribavirin reduces the risk of death in HFRS patients by up to 7% when given at an early stage, and is apparently inefficient in HPS patients (Mertz et al., 2006). A broad-spectrum pyrazine by-product, Favipiravir (5-fluoro-2-hydroxypyrazine-3- carboxamide), which is more tolerated in humans than Ribavirin is, has shown to improve the survival rate of hamsters infected with ANDV when taken daily. Perhaps the oldest antiviral treatment against diseases caused by hantaviruses consists in transferring serum from surviving patients to patients in the acute stage. This technique has been used by Russian, Chinese, and Chilean groups. The effect of immune serum in patients is thought to be based on the neutralization of the virus, which prevents cell entry.

This strategy seems especially efficient before and during the maximum virus release into the bloodstream. Particularly in HFRS or HPS cases, the concentration of viral particles in the bloodstream (viremia) reaches its maximum during the acute stage. In patients who are in the acute stage of the disease, passive transfer of neutralizing antibodies has been suggested as a way to reduce the viral load and thus extend survival time in the early stage, during which a protective immune response can develop.

Experiments with Syrian hamsters, the animal model for studying HPS, reinforce the idea that transferring neutralizing antibodies to HPS patients in the early stage could protect them from developing a severe form of this disease. These rodents received immune serum from Rhesus monkeys (Macaca mulatto) that were vaccinated with DNA encoding the Andes virus glycoproteins. After receiving the neutralizing antibodies 1-5 days after receiving a lethal dose of the Andes virus, the hamsters proved to have protection from the disease. Likewise, in the case of the Hantaan virus in rodents, neutralizing antibodies have shown to protect them from hantavirus infection. Similarly, administering immune serum to rodents before hantavirus infection, interferes with target cell infection, while administering it after infection, limits the infection of new target cells.

This data suggests that a treatment blocking hantavirus infection in cells could be effective in patients in a prophylactic sense and even during the early stage of the disease. Nonetheless, despite the efficiency of these antibodies in hamsters and rodents, giving them to humans requires humanizing them so that the patient does not have an immune response to rodent antibodies. Humanized antibodies against the hantavirus are not being used in China, probably due to their high production cost (personal communication, E.K. Bautz). As for HPS, an open clinical study on the efficacy of plasmapheresis in HPS patients in Chile was carried out from 2008 to 2012. 29 out of 32 patients were treated and reached a mortality rate of 14%, compared to a 32% rate for non-treated patients during the same period and 28% for non-treated patients in the same geographic zone (Vial et ah, 2015). Although this treatment seemed safe, this procedure is difficult to standardize since 1) the titer of neutralizing antibodies in humans varies over time and 2) vast quantities are required to guarantee high antibody concentrations in the patient during a reasonable time. Access to surviving patients' plasmas is generally scarce and is a variable that is difficult to control. The scarceness of immune sera is the reason why it is currently only used in patients with severe symptoms, and not at earlier time points as a post-exposure prophylaxis or preventive in persons that may face risk of exposure to infection. Overall, this data shows the need to develop antiviral medicaments that block hantavirus entry into cells and that can be produced in great quantities at a low cost. HANTAVIRUS BIOLOGY

Hantaviruses belong to the Bunyaviridae family and have a tripartite genome made up of negative- sense, single-stranded RNA. In September 2016, after the filing of the patent application priority of this case, the International Committee on Taxonomy of Viruses changed the classification of hantaviruses. Since this moment, a new order termed "Bunyavirales" was created, including at total of nine families. Among them, Hantaviruses form now an independent family, the Hantaviridae, which includes currently one genus termed Orthohantavirus. The other 7 families of the Bunyavirales order include 3 additional families that comprise human pathogenic viruses such as Crimean Congo Hemorrhagic Fever virus, Rift Valley Fever virus and La Crosse virus belonging respectively to the Nairoviridae (formerly Nairovirus genus), Phenuiviridae family (former Phlebovirus genus), and Peribunyaviridae (former Orthobunyavirus genus) (https://talk.ictvonline.org/taxonomy/). To avoid confusions, in this invention, we will continue using the former classification of the Bunyavirdae family (Elliott, 1990). In this sense, we will use the classification "Hantavirus genus" and "hantaviruses" and refer thereby to viruses of the new Orthohantavirus genus and other members in the Hantaviridae family.

The tripartide RNA genome of hantaviruses consists of the small segment (S) that encodes at least the nucleocapsid protein (N); the large segment (L) that encodes the RNA polymerase depending on RNA; and the medium segment (M) that encodes the glycoprotein precursor (GPC), which is processed co-translationally, producing the Gn and Gc glycoproteins (Elliott, 1990).

Hantavirus particles consist of ribonucleocapsids, which are made up of the nucleoprotein that covers the various genomic segments of viral RNA and the virus's RNA polymerase. These ribonucleocapsids are wrapped in a lipid membrane that hantaviruses acquire from cell membranes when exiting the cell. The Gn and Gc glycoproteins are anchored to this membrane, each through its own transmembrane region. These proteins are assumed to form heteromultimeric associations and probably mediate the process of virus entry into the cell, as has been demonstrated for many other viruses. Little is yet known about how hantaviruses and others from the Bunyaviridae family enter cells.

Cell entry is initiated through virus binding to cell receptors. Various entry factors have been identified for hantaviruses, among them αγβ ₃ integrins, decay- accelerating factors (DAF/CD55), the receptor for the globular head domain of complement Clq (gClqR/p32), and an unidentified protein of 70 kDa. After interacting with cell receptors, hantaviruses are uptaken by endocytosis. From the endosomes, the virus must release its ribonucleocapsids into the cell's cytoplasm in order to start replication. To this end, the ribonucleocapsids must cross the endosomal membrane barrier. This key step in cell infection occurs through the fusion of the virus membrane with the endosomal membrane in a low-pH environment.

VIRAL MEMBRANE FUSION PROTEINS

The membrane fusion process is induced by viral envelope proteins. So far, three different classes of fusion proteins have been described based on their structures. Class I fusion proteins are formed mainly by alfa helices and class II proteins by beta sheets, while class III proteins combine structural characteristics from class I and class II proteins (reviewed by Harrison, 2015). Regardless of their molecular structures, all fusion proteins contain a transmembrane region through which they are anchored to the viral envelope membrane. On the other side of the molecule, activated fusion proteins expose a fusion peptide that promotes their insertion into target membranes. Once in the membranes, fusion peptides are believed to destabilize the target membrane in favor of fusing the opposed membranes. Once membrane fusion concluded, fusion proteins are assumed to have changed their conformation multiple times, reaching a highly stable hairpin- like post- fusion conformation. In this structure, the fusion peptide is located juxtaposed to the transmembrane region, both inserted into the same membrane (reviewed by Kielian & Rey, 2006).

For hantaviruses, it has been experimentally demonstrated that the fusion activity is conferred by their glycoproteins, and we have associated it specifically with the Gc protein, which further seems to share features with class II fusion proteins (Tischler et al , 2005). The Gc protein has also been identified as a fusion protein in other members of the Bunyaviridae family, such as the La Crosse and Bunyamwera viruses, both from the Orthobunyavirus genus. As for phleboviruses, which are a separate genus in the Bunyaviridae family, they have recently been confirmed as having the structural characteristics of class II fusion proteins. It remains to be seen whether other members of the Bunyaviridae family have class II fusion proteins, especially since recent data on the Flaviviridae family has demonstrated that viruses of different genera in this family have substantial differences among their fusion proteins (Li & Modis, 2014). Class II fusion proteins have a characteristic ectodomain structure divided into 4 domains: domains I-III and the stem region connecting domain III with the transmembrane region (Harrison, 2015). By comparing class II fusion proteins, in silico studies allowed us to define amino acids that probably encompass each of these different domains within hantavirus Gc proteins (Tischler et al , 2005): domain I, which includes the Gc N-terminal; domain II, which includes a putative fusion peptide; and domain III (compare with Figures 1A and IB). Once we defined the putative domain III and predicted the sequence of the Gc transmembrane region, it was possible to identify the amino acid sequence of the putative hantavirus Gc stem region (data not shown). The structural models obtained in these in silico analyses were complemented by functional in vitro studies, where the essential role of the fusion peptide and the predicted stem region were demonstrated to correspond to functional regions of Gc of hantaviruses. INHIBITION OF VIRAL FUSION PROTEINS

It is well-known that the ligands that bind selectively to a conformation of viral fusion proteins can block or delay viral entry when this conformation precedes membrane fusion. As of today, two different strategies have been developed to inhibit fusion proteins and will be detailed here.

The first strategy consists of designing molecules that bind to small cavities in fusion proteins, which disappear during the transition from its pre-fusion to its post-fusion conformation. In the case of the human immunodeficiency virus (HIV; class I), molecules have been identified that bind to the surface of the internal helix bundle of the gp41 fusion protein. For the Dengue virus (class II), a cavity has been identified in a pocket located in the interphase of domains I and II; this region represents a hinge that allows domain II to rotate in relation to domains I and III. Multiple molecules that bind with high affinity to this pocket alter the balance of conformations; thereby inhibit membrane fusion and thus cell infection. Nevertheless, this strategy requires knowledge of the molecular structure of the fusion protein to be inhibited. For the fusion protein of hantaviruses and that other members of the Bunyaviridae family, except for phleboviruses, no structural information is yet available.

Another strategy that does not require information on the molecular structure of the fusion protein consists of using molecules that bind to regions involved in intramolecular interactions during the structural change of fusion proteins. Binding of an exogenous peptide, which is analogous to an interacting region from the external layer of a fusion protein, can prevent the post-fusion hairpin conformation required for membrane fusion, and thus inhibits viral entry into the cell.

For class I fusion proteins, the intramolecular interacting regions correspond to a region near the C-terminal (C-terminal heptad repeat), which interacts with a region near the N- terminal (N-terminal heptad repeat). Peptides containing C-terminal heptad repeat sequence bind to the three-helical bundle on the internal layer of the HIV gp41 protein, and thus prevent the formation of the stable post-fusion hairpin conformation. This strategy of inhibiting membrane fusion has been used successfully in humans with a peptide derived from HIV that is termed T20/ 'Enfuvirtide. This is a licensed drug corresponding to the C-terminal helix of HIV gp41.

In the post-fusion stage of class II fusion proteins, the intramolecular interacting regions on the external layer correspond to domain III and the stem region. For the fusion proteins of Dengue and Semliki viruses (class II), it has been shown that domain III, with or without the stem region, inhibits membrane fusion and thus is a functional analogue of the C- terminal heptad repeat regions in class I fusion proteins (Liao & Kielian, 2005). This inhibition seems to be based on the fact that recombinant domains III, with or without the stem region, bind to the central core of the fusion protein, formed by trimers of domains I and II. In this way, they impede the formation of the post-fusion hairpin of the protein and thus membrane fusion (Liao y Kielian, 2005). Therefore, molecules that imitate regions on the external layer of the hairpin conformation of the post-fusion trimer of class I and II viral fusion proteins are potent inhibitors of membrane fusion, and consequently of virus entry into the cell.

In a previous patent (CL 51057), we defined recombinant proteins of 100-144 residues, encompassing putative domain III with or without the putative stem region of the hantavirus Gc protein, which inhibit the membrane fusion process mediated by said Gc protein and thus block cell infection. In the current invention, we define Gc fragments of fewer than 25 residues, encompassing fragments of domain III or the stem region of the hantavirus Gc protein, which inhibit infection. As a result, these fragments have immense potential for preventing hantavirus infection in humans or for treating infected patients.

To date, the state of the art regarding invention patents shows a lack of technical solutions to infections by viruses from the Hantavirus genus, such as Andes, Puumala, and others. Such infections could be prevented and/or treated with the new peptides claimed in this invention.

Among previous patent documents that are related only tangentially to this technology, the following should be mentioned.

On the one hand, we have patent documents related to therapeutic and/or prophylactic approaches associated with using pharmaceutical drugs to treat viral infections. One example is the North American patent published as US2014/328792, which describes useful methods and compounds to treat and prevent viral infections, specifically using squalamine and its derivates.

On the other hand, we have patent documents related to therapeutic and/or prophylactic approaches that use immunogenic agents and vaccines producing an immunologic antiviral response in the treated individual, such as the PCT patent application published as WO2010/037402. This patent reports on a technology used for developing multimeric antigens that help to create antiviral vaccines.

SUMMARY OF THE INVENTION

In order to infect a cell, an enveloped virus must binds to cell receptors and later fuse its membrane with that of the host cell. Our seeking to prevent cell infection by hantavirus has demonstrated that there are fragments derived from the Gc protein of the Hantavirus genus in the Bunyaviridae family that inhibit the fusion process. These fragments, which are already patent-protected (CL 51057) and longer than 100 amino acids, differ from those proposed in this application, which correspond to shorter sequences of fewer than 25 amino acids and unexpectedly still have an efficient inhibitory effect.

The advantage of using fragments from domain III and the stem region instead of the complete sequences of these domains is the greater ease with which the former can be synthesized. This lowers the production cost without losing efficiency in virus inhibition. In addition, these short fragments do not produce an adaptive immune response and thus have the advantage that, unlike the complete domains, they can be administered repeatedly to a eukaryotic organism, preferably a mammal, without provoking undesirable immune reactions.

In this context, the present invention refers in general to Gc protein fragments from the Hantavirus genus that inhibit infection of eukaryotic cells by hantaviruses inhibiting membrane fusion.

Furthermore, this invention refers to using fragments of the Gc protein of the Hantavirus genus to prevent infection and to be used in treating infected patients. The invention also defines pharmaceutical compounds that contain said fragments, which are useful for impeding cell infection by hantavirus as well as for producing said compound. In one particular embodiment, the invention refers to fragments of the Gc protein of the Hantavirus genus that preferably comprise SEQ ID NOs: 1 and 2, located in the putative domain III

In another embodiment, the invention refers to additional fragments of the Gc protein of the Hantavirus genus, preferably those with SEQ ID NOs: 3, 4, and 5, located in the putative stem region. The preferred embodiments for all sequences SEQ ID NOs: 1 , 2, 3, 4, and 5, showed inhibition of the membrane fusion process associated with cell infection by hantaviruses. On a preferred embodiment level, sequences SEQ ID NOs: 1 , 2, 3, 4, and/or 5 are especially efficient at inhibiting infection by the Andes virus. In said embodiment, the hantavirus Gc protein is represented by the amino acid sequence SEQ ID NO: 74.

Besides inhibiting the Andes virus, the fragments comprising sequences 1 , 2, 3, 4, and/or 5 also inhibit other phylogenetically distant hantaviruses, such as the Puumala virus. In view of this unexpected cross reaction, a common pattern was identified for each sequence (SEQ ID NOs: 1 , 2, 3, 4, and 5) of the Gc glycoprotein of Andes virus and analogous sequences in the Gc glycoprotein of Puumala virus (SEQ ID NOs: 6, 7, 8, 9, and 10). As a result, we were able to identify the following sequence patterns, where XI is an apolar amino acid, X2 is a polar amino acid, X3 is a negatively charged amino acid, X4 is a positively charged amino acid, and X5 is any amino acid:

The pattern shared by SEQ ID NO: 1 of Andes virus Gc protein (peptide Dili IA1) and the analogous Puumala virus SEQ ID NO: 6 is: X ₃ GAWGSGVGFX ₂ LX ₅ ;

The pattern shared by SEQ ID NO: 2 of the Andes virus Gc protein (peptide Dili IA2) and the analogous Puumala virus SEQ ID NO: 7 is: X ₁ RGX ₂ NTX ₁ X ₄ VVGK;

The pattern shared by SEQ ID NO: 3 of Andes virus Gc protein (peptide R2) and the analogous Puumala virus SEQ ID NO: 8 is: XjXsXsC FXsKXsGE XiLGXiLNGN; The pattern shared by SEQ ID NO: 4 of the Andes virus Gc protein (peptide R2.1) and the analogous Puumala virus SEQ ID NO: 9 is: XsXsXsCWFXsKXsG;

The pattern shared by SEQ ID NO: 5 of the Andes virus Gc protein (peptide R2.2) and the analogous Puumala virus SEQ ID NO: 10 is: EWXiLGXjLNGN.

Given the significant phylogenetic distance between the Andes hantavirus and the

Puumala hantavirus, the question raised was whether the five sequence patterns identified were also present in other sequences of the Gc glycoprotein of hantaviruses that are available in databases. Surprisingly, the five patterns were found in other hantaviruses. Because of this, the invention is extended also to other Gc fragments different from those of Andes and Puumala viruses, whose sequence pattern can be found in hantaviruses.

Based on the sequence patterns shared by the Andes and Puumala viruses, we identified equivalent regions in other viruses of the Hantavirus genus whose Gc glycoprotein sequences are currently known.

Pattern X ₃ GAWGSGVGFX ₂ LX ₅ (peptide Dili IA1) was found in the following hantaviruses:

- Bermejo, Cano Delgadito, Choclo, Dobrava-Belgrade, Hantaan, Lechiguanas,

Maciel, Maporal, Oran, Pergamino, Rio Mamore, Seoul, Castelo dos Sonhos virus, Gou, Necocli (SEQ ID NO: 11),

Khabarovsk, Muju, Prospect Hill, Tula, Hokkaido,

Yuanjiang (SEQ ID NO: 12),

- Araraquara, Bayou, Black Creek Canal, El Moro Canyon, Laguna Negra,

Catacamas, Limestone Canyon, Montano (SEQ ID NO: 13),

New York, Sin Nombre, Monongahela-2 (SEQ ID NO: 14),

Sangassou (SEQ ID NO: 15),

Topografov (SEQ ID NO: 16),

- Cao y Oxbow (SEQ ID NO: 17), Fusong-Mf-682 (SEQ ID NO: 18),

hantavirus Human/HRP/02-72/BRA/2002, hantavirus

CGRn8316, hantavirus CGRn9415, hantavirus Jurong

TJK/06(RT49), hantavirus AH09, hantavirus Z10, hantavirus Liu, Soochong (SEQ ID NO: 19).

Pattern X 1 RGX2NTX 1 X4 VVGK (peptide Dili IA2) was found in the following hantaviruses:

Araraquara, Laguna Negra, Lechiguanas, Maporal, Oran, hantavirus Human/HRP/02-72/BRA/2002 (SEQ ID NO: 20),

Khabarovsk (SEQ ID NO: 21),

Bayou (SEQ ID NO: 22),

Bermejo (SEQ ID NO: 23),

Catacamas (SEQ ID NO: 24),

Muju, Tula y Yuanjiang (SEQ ID NO: 25),

El Moro Canyon (SEQ ID NO: 26),

New York, Sin Nombre, Monongahela-2 y Necocli (SEQ ID NO: 27),

Topografov (SEQ ID NO: 28),

Castelo dos Sonhos (SEQ ID NO: 29),

Fusong-Mf-682 (SEQ ID NO: 30).

Pattern X5X5X5CWFX5KX5GEWX1LGX1LNGN (peptide R2) was found in the following hantaviruses:

Lechiguanas, Rio Mamore (SEQ ID NO: 31),

Araraquara (SEQ ID NO: 32),

Black Creek Canal (SEQ ID NO: 33), Laguna Negra, Human/HRP/02-72/BRA/2002 (SEQ ID NO: 34) Montano (SEQ ID NO: 35),

New York, Sin Nombre, Limestone Canyon (SEQ ID NO: 36),

Oran (SEQ ID NO: 37),

- Castelo dos Sonhos (SEQ ID NO: 38),

Hokkaido (SEQ ID NO: 39),

Monongahela-2 (SEQ ID NO: 40).

Pattern X5X5X5CWFX5KX5G (peptide R2.1) was found in the following hantaviiuses: - Lechiguanas, Rio Mamore, Castelo dos Sonhos (SEQ ID NO: 41),

Araraquara, Necocli (SEQ ID NO: 42),

Bayou, Catacamas (SEQ ID NO: 43),

Black Creek Canal (SEQ ID NO: 44),

Cano Delgadito (SEQ ID NO: 45),

- Choclo (SEQ ID NO: 46),

El Moro Canyon (SEQ ID NO: 47),

Dobrava-Belgrade (SEQ ID NO: 48),

Hantaan, hantavirus AH09, hantavirus Liu, Rockport, Soochong (SEQ ID NO: 49),

- Khabarovsk, Seoul, Yuanjiang (SEQ ID NO: 50)

Laguna Negra, hantavirus Human/HRP/02-72/BRA/2002 (SEQ ID NO: 51), Maporal (SEQ ID NO: 52),

Muju (SEQ ID NO: 53),

New York, Sin Nombre, Limestone Canyon (SEQ ID NO: 54),

- Oran (SEQ ID NO: 55), Prospect Hill, Topografov, Tula (SEQ ID NO: 56),

Sangassou (SEQ ID NO: 57),

Thailand 741 (SEQ ID NO: 58),

Asama (SEQ ID NO: 59),

- Cao (SEQ ID NO: 60),

Fusong-Mf-682, Gou, hantavirus CGRn9415 (SEQ ID NO: 61),

hantavirus CGRn8316 (SEQ ID NO: 62),

hantavirus Z10 (SEQ ID NO: 63),

Hokkaido (SEQ ID NO: 64),

- Jurong TJK/06(RT49) (SEQ ID NO: 65),

Montano (SEQ ID NO: 66),

Monongahela-2 (SEQ ID NO: 67),

Oxbow (SEQ ID NO: 68). Pattern EWXILGXILNGN (peptide R2.2) was found in the following hantaviruses:

Araraquara, Black Creek Canal, Laguna Negra, Lechiguanas, New York, Oran, Rio Mamore, Sin Nombre, Montano, Monongahela-2 (SEQ ID NO: 69),

Hokkaido (SEQ ID NO: 70)

- Castelo dos Sonhos. (SEQ ID NO: 71)

hantavirus Human/HRP/02-72/BRA/2002, Limestone Canyon (SEQ ID NO: 72).

Sequences SEQ ID NOs: 1-72 can contain amino acid sequences at their N- and/or C-terminal that facilitate their solubility in an aqueous medium and interphase with membranes, as well as their purification through affinity columns. This sequence may include, for example, 6 histidines. Alternatively, SEQ ID NOs: 1-72 that are synthesized without an additional sequence at their N-terminal may contain a methionine insert at their N-terminal as a way to obtain the recombinant protein.

Sequences SEQ ID NOs: 1-72 can contain insertions of simple or multiple amino acids. Preferably, these modifications are introduced at the N-terminal and/or C-terminal ends.

Sequences SEQ ID NOs: 1-72 can contain an N-terminal and a C-terminal that vary in length from 1 to 4 additional amino acids. In a particular embodiment, the N- or C- terminal of sequences SEQ ID NOs: 1-72 may vary regarding to their sequences in the Gc protein. In this context, SEQ ID NOs: 1-72 can also be 1-4 amino acids shorter at their N- and/or C-terminal ends.

Sequences SEQ ID NOs: 1 -72 may contain deletions, mutations, and insertions that imply the elimination, mutation, or insertion of one or more amino acids.

Sequences SEQ ID NOs: 1-72 may contain moieties, useful for cell targeting or improving in vivo stability, which can include chemical, biochemical and/or physical modifications. Such modifications can include but are not limited to, amino acids, fatty acids, lipids, steroid, sugar, carbohydrate, nucleic acids and/or synthetic compounds. Such a modification can be covalently linked to the Gc fragments by chemical groups including amino acid, amine, carboxyl, thiol, sulfhydryl, or disulfide groups. Modifications also cover other types of chemical bonds and conjugations.

In a particular embodiment, such a moiety corresponds to any modification that enhances association of the Gc fragments with the cell membrane and/or its association with an intracellular compartment such as, but not limited to, endosomes. In another particular embodiment, such targeting signal corresponds to amino acid modifications at the N-/or C- terminal of the Gc fragments including the sequence RGD (arginine-glycine-aspartic acid). In another particular embodiment, the modification of the Gc fragments at their N-/or C-terminal corresponds to a lipid, still more particularly to cholesterol, stearic acid, decanoic acid and/or cyteamine. In another particular embodiment, the modification of the Gc fragments comprises a carbohydrate, and still more particularly, galactose residues.

This invention also refers to Gc fragments that are useful for inhibiting the infection of eukaryotic cells by hantaviruses. These cells include those derived from mammals, such as African green monkey cells {Chlorocebus sp.) or human cells. Preferably, the cells should be part of a multicellular eukaryotic organism, especially a mammal and, preferentially, a human, which is susceptible to hantavirus infection, or which is at risk of hantavirus infection, or which is infected by hantaviruses.

In another embodiment, the invention refers to a prophylactic and/or therapeutic method, which comprises the use of the Gc fragments containing at least one sequence selected from a group consisting of SEQ ID NOs: 1 -72. The Gc fragments used for preventive or therapeutic intervention, may containing modifications in the sequence selected from a group of SEQ ID NOs: 1 -72, that can include chemical, biochemical and/or physical modifications, such as amino acids, fatty acids, lipids, steroid, sugar, carbohydrate, nucleic acids and/or synthetic compounds.

These Gc fragments can be used in any pharmacological acceptable concentration that prevent infection by hantaviruses or produces de inhibition of hantavirus infection.

The fragments can be included in pharmacological compositions to be administered to mammals, including humans. The dose can be determined by standard protocols for dose response, which are of common knowledge for experts in the field.

The compositions may contain vehicles and/or acceptable pharmacological excipients, which will vary depending on the pharmaceutical form that is desired to be prepared, as well as the dose and route of administration. The pharmaceutical composition to be used in a preventive or therapeutic manner can be administrated by any of the routes generally used in the field. Such a pharmacological composition may be formulated to be delivered by parenteral, oral or topical routes, including intravenous, intradermal, intramuscular, intraperitoneal, nasal, oral, subcutaneous or epicutaneous routes. In a preferred embodiment of the present invention, the pharmacological composition may be formulated to be delivered through the respiratory route by inhalation, as well as through the intravenous route, e.g. by injection.

Due to the high structural and functional conservation of the hantavirus Gc fusion protein, it is expected that the pharmaceutical composition is useful in preventing and treating the infection by viruses from the Hantavirus genus. These include classified hantaviruses such as the Andes virus, the Araraquara virus, the Bayou virus, the Bermejo virus, the Black Creek Canal virus, the Cano Delgadito virus, the Choclo virus, the Dobrava-Belgrade virus, the El Moro Canyon virus, the Hantaan virus, the Khabarovsk virus, the Laguna Negra virus, the Lechiguanas virus, the Maciel virus, the Maporal virus, the Muju virus, the New York virus, the Oran virus, the Pergamino virus, the Prospect Hill virus, the Puumala virus, the Rio Mamore virus, the Sangassou virus, the Seoul virus, the Sin Nombre virus, the Topografov virus, and the Tula virus. This genus also includes unclassified hantaviruses, such as the Asama virus, the Catacamas virus, the Cao virus, the Castelo dos Sonhos virus, the Gou virus, the Hokkaido virus, the Fusong-Mf-682 virus, the Limestone Canyon virus, the human/hrp/02-72/bra/2002 hantavirus, hantavirus CGRn8316, hantavirus CGRn9415, hantavirus Jurong, hantavirus AH09, hantavirus Z10, hantavirus Liu, the Montano virus, the Monongahela-2 virus, the Necocli virus, the Oxbow virus, the Rockport virus, the Soochong virus, and the Yuanjiang virus. This classification is in line to the established classification by the International Committee on Taxonomy of Viruses, at the moment of the priority date invoked for this invention. In preferred embodiments, the Gc fragments and/or the pharmaceutical composition can be used in vitro (e.g. cell culture) or in vivo, preferably administering them to a living eukaryotic organism. Preferably, the eukaryotic organism is a mammal, and still more preferably, the organism corresponds to a human.

In a preferred embodiment the invention uses any of the fragments described above because they enable the preparation of a medicament to prevent or treat hantavirus infections.

All the peptides that were used, were first isolated from domain III and from the stem region by recombinant synthesis in Escherichia coli. Given their short sequences, these peptides were later produced by chemical synthesis.

The invention includes a procedure to synthesize Gc protein fragments from the Hantavirus genus. This synthesis procedure can follow any of the methods known in the field or a combination of them, such as biosynthesis, synthesis by recombinant expression, enzymatic synthesis, chemical synthesis, synthesis by chemical peptide condensation, synthesis by polypeptide digestion, synthesis of peptides in the liquid or solid phase, or any other phase known in the field, peptide synthesis on a small, medium, or large scale, microwave-assisted peptide synthesis, or any other ad hoc method.

Additionally, the present invention comprises useful kits for prophylactic and/or therapeutic treatment of the infection of cells by viruses from the Hantavirus genus. In another embodiment, the invention also could comprise diagnostic and/or prognostic kits for the detection of hantavirus infections. In a particular embodiment, the infection may be produced by the Andes and/or Puumala hantaviruses and still more particularly of Andes virus. These kits comprise at least one of the sequences disclosed in this invention. Of particular interest are the kits comprising at least one of the peptides defined by any of the following sequences: SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72.

In a particular embodiment, the invention refers to a fragment of the Gc fusion protein from the Hantavirus genus that inhibits membrane fusion at least between one type of hantavirus and the membrane of a eukaryotic cell, wherein the fragment is defined by a polypeptide selected from any of the following sequences:

a) X3GAWGSGVGFX2LX5 located in the putative domain III of Gc; and/or b) X ₁ RGX ₂ NTX ₁ X ₄ VVGK located in the putative domain III of Gc; and/or c) XsXsXsCWFXsKXsGEWXiLGXiLNGN located in the putative stem region of Gc; and/or

d) X ₅ X ₅ X ₅ CWFX ₅ KX ₅ G located in the putative stem region of Gc; and/or e) EWX ₁ LGX ₁ LNGN located in the putative stem region of Gc;

wherein Xi is an apolar amino acid, X ₂ is a polar amino acid, X ₃ is a negatively charged amino acid, X ₄ is a positively charged amino acid, and X ₅ is any amino acid.

In another embodiment of the invention, the fragment derived from the putative domain III or the putative stem region is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72. In a preferred embodiment, the fragment, is selected from the group consisting of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, wherein said sequences are comprised by the putative domain III.

In a more preferred embodiment, the fragment is selected from the group consisting of SEQ ID NOs: 1, 6, 11, 12, 13, 14, 15, 16, 17, 18, and 19, wherein said sequences are comprised by the putative domain III and follows the pattern X ₃ GAWGSGVGFX ₂ LX _5, located in the putative domain Illof Gc. In a particular embodiment, the fragment is selected from the group consisting of SEQ ID NOs: 1 and 6, wherein said sequences are comprised by the putative domain III and follows the pattern X ₃ GAWGSGVGFX ₂ LX ₅ . In the most preferred embodiment the fragment is defined by the sequence of SEQ ID NO: 1 , wherein said sequence is comprised by the putative domain III and follows the pattern X ₃ GAWGSGVGFX ₂ LX _5.

In another embodiment the fragment is selected from a the group consisting of SEQ ID NOs: 2, 7, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, wherein said sequences are comprised by the putative domain III and follow the pattern X ₁ RGX ₂ NTX ₁ X ₄ VVGK, which is located in the putative domain III of Gc. In a more preferred embodiment the fragment is selected from the group consisting of SEQ ID NOs: 2 and 7, which are comprised in the putative domain III and follow the pattern X ₁ RGX ₂ NTX ₁ X ₄ VVGK. In the most preferred embodiment the fragment is defined by the sequence of SEQ ID NO: 2, which is comprised by the putative domain III and follows the pattern X ₁ RGX ₂ NTX ₁ X ₄ VVGK.

In a further embodiment, the fragment is selected from a the group consisting of SEQ ID NOs: 3, 4, 5, 8, 9, 10, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72, wherein said sequences are comprised by the putative stem region. According to an additional embodiment, the fragment is selected from the group consisting of SEQ ID NOs: 3, 8, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, wherein said sequences are comprised by the putative stem region and follow the pattern X ₅ X ₅ X ₅ CWFX ₅ KX ₅ GEWX ₁ LGX ₁ LNGN, which is located in the putative stem region of Gc. In a preferred embodiment the fragment is selected from the group consisting of SEQ ID NOs: 3 and 8, wherein said sequences are comprised by the putative stem region and follow the pattern In a most preferred embodiment the fragment is defined by SEQ ID: 3, wherein said sequence is comprised by the putative stem region and follows the pattern X ₅ X ₅ X ₅ CWFX ₅ KX ₅ GEWX ₁ LGX ₁ LNGN. In an additional embodiment, the fragment, is selected from the group consisting of SEQ ID NOs: 4, 9, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, wherein said sequences are comprised by the putative stem region and follow the pattern X ₅ X ₅ X ₅ CWFX ₅ KX ₅ G, which is located in the putative stem region of Gc. In a preferred embodiment, the fragment, is selected from the group consisting of SEQ ID NOs: 4 y 9, wherein said sequences are comprised by the putative stem region and follow the pattern X ₅ X ₅ X ₅ CWFX ₅ KX ₅ G. In a most preferred embodiment the fragment is defined by SEQ ID NO: 4, wherein said sequence is comprised by the putative stem region and follows the pattern X ₅ X ₅ X ₅ CWFX ₅ KX ₅ G.

In another embodiment, the fragment is selected from the group consisting of

SEQ ID NOs: 5, 10, 69, 70, 71 , and 72, wherein said sequences are comprised by the putative stem region and follow the pattern EWX ₁ LGX ₁ LNGN. In a preferred embodiment the fragment, is selected from the sequence group consisting of SEQ ID NOs: 5 and 10, wherein said sequences are comprised by the putative stem region and follow the pattern EWX ₁ LGX ₁ LNGN. In a more preferred embodiment the fragment is defined by SEQ ID NO: 5, wherein said sequence is comprised by the putative stem region and follows the pattern EWX ₁ LGX ₁ LNGN.

In certain embodiment the fragment is capable of inhibiting the fusion of the envelope membrane of Andes virus and/or Puumala virus with a membrane of at least one eukaryotic cell. In another embodiment the fragment is capable of inhibiting the fusion of the Andes virus envelope membrane with a membrane of at least one eukaryotic cell. In another particular embodiment the fragment is capable of inhibiting the fusion of the Puumala virus envelope membrane with a membrane of at least one eukaryotic cell.

In an additional embodiment, the invention refers to a pharmaceutical composition, wherein said composition comprises a fragment of the Gc fusion protein of the Hantavirus genus, able to inhibit the fusion of said virus with a eukaryotic cell, together with a vehicle and/or acceptable pharmaceutical excipient.

In a particular embodiment the invention relates to a method for inhibiting the fusion of a hantavirus with a cell of a eukaryotic organism, comprising the administration of a fragment of the Gc fusion protein of the Hantavirus genus according to any of the above described embodiments, which is useful to prevent or treat hantavirus infection of said eukaryotic organism.

In another particular embodiment, the invention refers to a method for inhibiting the fusion of a hantavirus with a cell of a eukaryotic organism, comprising the administration of the pharmaceutical composition defined herein, which is useful to prevent or treat hantavirus infection of said eukaryotic organism.

In an additional embodiment, the invention refers to a method of preventing, treating and/or inhibiting hantavirus infection of a eukaryotic organism, comprising the administration of a pharmacological acceptable concentration of at least one of the fragments defined herein, or of a pharmaceutical composition defined herein, where said eukaryotic organism is in need thereof. In a preferred embodiment said eukaryotic organism is an animal. In a further preferred embodiment said animal is a mammal. In a more preferred embodiment said mammal is a human. In certain preferred embodiments, the eukaryotic organism is:

a. susceptible to hantavirus infection; and/or

b. at risk of hantavirus infection; and/or

c. infected by hantaviruses.

In certain embodiment, the treatment or preventing method produces cross protection against at least the Andes and Puumala hantaviruses. In an additional embodiment, the invention refers to a therapeutic kit comprising at least one of the fragments, or a pharmaceutical composition as defined herein, wherein said kit is useful for being used in the prevention and/or treatment of an hantavirus infection.

DEFINITIONS VLP: The term VLP is an abbreviation for virus-like particle. Hantavirus VLPs are viral particles that resemble those of the native hantaviruses in both, structural and antigenic terms. This type of particle consists of a lipid bilayer membrane in which Gn/Gc glycoproteins are anchored. The VLPs used here lack other viral proteins and viral RNA, and were prepared as described in Chilean patent application CL01085-2011 : by expressing viral Gn/Gc glycoproteins in 293FT cells and purifying them from the supernatant of transfected cells by ultracentrifugation Mock: This term refers to an experimental condition in which there is no inhibitor, such as for example the cell infection by the Andes virus or the fusion activity among cells.

Peptide NN: This peptide, consisting in 42 amino acids, has sequence SEQ ID NO: 73 and is derived from the sequence of the Andes virus nucleoprotein. This peptide was used as a negative control in all the embodiment examples since it is well-known that the nucleoprotein does not influence the fusion activity of the Andes virus.

Syncytium: This is a cell containing several nuclei and is the product of plasma membrane fusion among various cells. The nuclei can be identified by DNA staining using, for example, the DAPI fluorophore (4',6-diamino-2-phenylindole), as it is well-known in the field. Fusion activity is expressed by the fusion index (FI), which is calculated according to the following formula:

IF = 1 - (number of cells/number of nuclei).

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1: Localization of peptides derived from domain III (Dili) and the stem region (S) of the hantavirus Gc protein. A) Molecular model for the structure of the Gc protein and its organization in different domains derived from Tischler et ah, 2005. Domains I and II (DI-DII) are indicated in gray and domain III in black. B) Diagram of the primary structure of Gc, indicating predicted residues for domain III (black) and the predicted stem region (gray). DI-DII indicates domains I-II, Dili indicates domain III, S indicates the stem region, TM indicates the transmembrane region, Ct indicates the cytoplasmic tail. C) Conservation of the sequence of Dili and the stem region of Gc from the Hantavirus genus, indicated by lining up multiple sequences represented by Logo. The conservation of each position is indicated by the total height of each pile in this position, while the conservation of specific residues is given in each position by the height of the symbols. For the multiple alignment, Gc glycoprotein sequences were downloaded from GenBank and represent the phylogroups established for hantaviruses from different reservoirs, including hantaviruses persisting in rodents from the following subfamilies: Avicolinae (PUUV; accession number NP_941983.1), Murinae (Hantaan virus; accession number NP_941978.1), Sigmodontinae (Andes virus; accession number AA086638.1)and also hantaviruses persisting in shrews, moles, and bats from the following families: Soricidae (Thottapalayam virus; access number ABU82619.1), Talpidae (Asama virus; access number ACI28508.1), and Rhinolophidae (Longquan virus; access number AGI62348.1). D) Localization of the regions encompassed by peptides Dili IA1, Dili IA2, R2, R2.1, and R2.2, in domain III and the stem region. Under each peptide, the sequence pattern shared by the Andes and Puumala viruses is shown.

Analysis of cytotoxicity in vitro generated by peptides derived from ANDV Gc. Vero E6 cells were incubated with peptides of ANDV Gc at two different concentrations during 18 hrs. Furthermore, the NN peptide derived from the nucleoprotein was used as a negative control. The Mock condition indicates the incubation of cells in absence of peptides. Inhibition of cell infection by ANDV using peptides of ANDV Gc. ANDV was co- incubated with different peptides during viral adsorption at different concentrations. NN was used as a negative control peptide. Mock corresponds to the incubation of ANDV with cells in the absence of peptides. Viral infection was detected by using flow cytometry, which detects the expression of the ANDV N protein.

Inhibition of syncytium formation by peptides derived from ANDV Gc. A) Inhibition of syncytium formation mediated by ANDV glycoproteins and B) cross inhibition of syncytium formation mediated by PUUV glycoproteins. Vero E6 cells expressing ANDV or PUUV glycoproteins were incubated during 5 min in a medium adjusted to pH 5.5, in the presence of different peptides of ANDV Gc or with the NN negative control peptide. Subsequently, the medium containing inhibitor candidates was substituted with neutral pH media and the cells were incubated during 4 h at 37°C. The fusion index is indicated as percentage in relation to the condition without peptide incubation (Mock). The NN peptide was used as a negative control. The fusion index (FI) was calculated with the following formula:

FI = 1 - (number of cells/number of nuclei).

Inhibition of ANDV fusion with the plasma membrane of Vero E6 cells. ANDV was pre-adsorbed for 1 h to Vero E6 cells at 4°C. The culture medium was later replaced by an E-MEM medium adjusted to pH 5.5 including the different peptides, and was incubated for 5 min at 37°C. Subsequently, the media including the peptides was replaced by E-MEM medium adjusted to pH 7.4, and cell infection by ANDV was allowed to proceed for 16h. Infected cells were quantified by the detection of the ANDV N protein expression using flow cytometry.

Gc fragment peptides impede the formation of a stable Gc post-fusion trimer. A) Multimerization state of Gc derived from ANDV VLPs in the presence and absence of peptide R2. ANDV VLPs were incubated at pH 7.0 or pH 5.5 in the presence and absence of the R2 peptide. Next, the oligomeric state of Gc was determined by saccharose gradient sedimentation. The presence of Gc in each fraction was analyzed by Western blot using a monoclonal anti-Gc antibody. B) Resistance of Gc to trypsin in the presence and absence of the R2 peptide. The ANDV VLPs were incubated for 30 min at different pHs in the presence and absence of the R2 peptide. Subsequently, TCPK trypsin was added and incubated for additional 30 min. The presence of Gc in each condition was detected by western blot. Results represent n=3 independent experiments. The statistical significance was evaluated using a Student's t-test of at least n=3 independent experiments and is shown in the figures using the following symbols: ***, P < 0,00025; **, P < 0,0025 ; *, P < 0,025.

BRIEF DESCRIPTION OF THE SEQUENCE LIST

Below is a listing of sequences of Gc fragments of the genus Hantavirus that are involved in the inhibition of hantaviruses.

The SEQ ID NO: 1 corresponds to a fragment of the partial, putative domain III of the Andes virus Gc protein termed Dili IA1.

The SEQ ID NO: 2 corresponds to another fragment of the partial, putative domain III of the

Andes virus Gc protein termed Dili IA2.

The SEQ ID NO: 3 corresponds to a fragment of the partial, putative stem region of the Andes virus Gc protein termed R2.

The SEQ ID NO: 4 corresponds to a fragment of the SEQ ID NO: 3 of the Andes virus Gc protein termed R2.1.

The SEQ ID NO: 5 corresponds to a fragment of the SEQ ID NO: 3 of the Andes virus Gc protein termed R2.2. The SEQ ID NO: 6 corresponds to a fragment of the putative domain III of the Puumala virus Gc protein equivalent to the SEQ ID NO: 1 of Andes virus.

The SEQ ID NO: 7 corresponds to another fragment of the putative domain III of the Puumala virus Gc protein equivalent to SEQ ID NO: 2 of Andes virus.

The SEQ ID NO: 8 corresponds to a fragment of the putative stem region of the Puumala virus

Gc protein equivalent to SEQ ID NO: 3 of Andes virus.

The SEQ ID NO: 9 corresponds to a fragment of the putative stem region of the Puumala virus

Gc protein equivalent to SEQ ID NO: 4 of Andes virus.

The SEQ ID NO: 10 corresponds to a fragment of the putative stem region of the Puumala virus

Gc protein equivalent to SEQ ID NO: 5 of Andes virus.

Based on conserved sequence patterns between the Andes virus and Puumala virus, equivalent regions were identified in other viruses of the Hantavirus genus whose sequences of the Gc glycoprotein are currently reported.

The SEQ ID NO: 11 is identical to the reference sequence SEQ ID NO: 1 of Andes virus and is also found in the hantaviruses: Bermejo, Cano Delgadito, Choclo, Dobrava-Belgrade, Hantaan, Lechiguanas, Maciel, Maporal, Oran, Pergamino, Rio Mamore, Seoul, Castelo dos Sonhos, Gou, Necocli.

The SEQ ID NO: 12 is identical to the reference sequence SEQ ID NO: 6 of Puumala virus and is also found in the hantaviruses: Khabarovsk, Muju, Prospect Hill, Tula, Hokkaido, Yuanjiang.

The SEQ ID NO: 13 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and SEQ ID NO: 6 of Puumala virus and is also found in the hantaviruses: Araraquara, Bayou, Black Creek Canal, El Moro Canyon, Laguna Negra, Catacamas, Limestone Canyon, Montano. The SEQ ID NO: 14 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and

SEQ ID NO: 6 of Puumala virus and is also found in the hantaviruses: New York, Sin

Nombre and Monongahela-2.

The SEQ ID NO: 15 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and SEQ ID NO: 6 of Puumala virus and is also found in the Sangassou hantavirus.

The SEQ ID NO: 16 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and

SEQ ID NO: 6 of Puumala virus and is also found in the Topografov hantavirus.

The SEQ ID NO: 17 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and

SEQ ID NO: 6 of Puumala virus and is also found in the hantaviruses: Cao and Oxbow. The SEQ ID NO: 18 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and

SEQ ID NO: 6 of Puumala virus and is also found in the Fusong-Mf-682 hantavirus.

The SEQ ID NO: 19 is equivalent to the reference sequences SEQ ID NO: 1 of Andes virus and

SEQ ID NO: 6 of Puumala virus and is also found in the hantaviruses: Human/HRP/02-

72/BRA/2002, CGRn8316, CGRn9415, Jurong TJK/06(RT49), AH09. Z10. Liu and Soochong.

The SEQ ID NO: 20 is identical to the reference sequence SEQ ID NO: 2 of Andes virus and is also found in the hantaviruses: Araraquara, Laguna Negra, Lechiguanas, Maporal, Oran, and Human HRP/02-72 BRA/2002.

The SEQ ID NO: 21 is identical to the reference sequence SEQ ID NO: 7 of Puumala virus and is also found in the Khabarovsk hantavirus.

The SEQ ID NO: 22 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the Bayou hantavirus.

The SEQ ID NO: 23 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the Bermejo hantavirus. The SEQ ID NO: 24 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and SEQ ID NO: 7 of Puumala virus and is also found in the Catacamas hantavirus.

The SEQ ID NO: 25 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and SEQ ID NO: 7 of Puumala virus and is also found in the hantaviruses: Muju, Tula and Yuanjiang.

The SEQ ID NO: 26 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the El Moro Canyon hantavirus. The SEQ ID NO: 27 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the hantaviruses: New York, Sin

Nombre, Monongahela-2 and Necocli.

The SEQ ID NO: 28 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the Topografov hantavirus.

The SEQ ID NO: 29 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the Castelo dos Sonhos hantavirus. The SEQ ID NO: 30 is equivalent to the reference sequences SEQ ID NO: 2 of Andes virus and

SEQ ID NO: 7 of Puumala virus and is also found in the Fusong-Mf-682 hantavirus.

The SEQ ID NO: 31 is identical to the reference sequence SEQ ID NO: 3 of Andes virus and is also found in the hantaviruses: Lechiguanas and Rio Mamore.

The SEQ ID NO: 32 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Araraquara hantavirus.

The SEQ ID NO: 33 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Black Creek Canal hantavirus. The SEQ ID NO: 34 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the hantaviruses: Laguna Negra and

Hu man/HRP/02 -72 B R A/2002. The SEQ ID NO: 35 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Montano hantavirus.

The SEQ ID NO: 36 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the hantaviruses: New York, Sin Nombre, Limestone Canyon.

The SEQ ID NO: 37 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Oran hantavirus.

The SEQ ID NO: 38 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Castelo dos Sonhos hantavirus. The SEQ ID NO: 39 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Hokkaido hantavirus.

The SEQ ID NO: 40 is equivalent to the reference sequences SEQ ID NO: 3 of Andes virus and

SEQ ID NO: 8 of Puumala virus and is also found in the Monongahela-2 hantavirus.

The SEQ ID NO: 41 is identical to the reference sequence SEQ ID NO: 4 of Andes virus and is also found in the hantaviruses: Lechiguanas and Rio Mamore and Castelo dos Sonhos.

The SEQ ID NO: 42 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Araraquara and

Necocli.

The SEQ ID NO: 43 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Bayou and

Catacamas.

The SEQ ID NO: 44 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Black Creek Canal hantavirus. The SEQ ID NO: 45 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the Cano Delgadito hantavirus. The SEQ ID NO: 46 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Choclo hantavirus.

The SEQ ID NO: 47 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the El Moro Canyon hantavirus. The SEQ ID NO: 48 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Dobrava-Belgrade hantavirus. The SEQ ID NO: 49 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Hantaan, AH09,

Liu, Rockport, Soochong.

The SEQ ID NO: 50 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Khabarovsk,

Seoul and Yuanjiang.

The SEQ ID NO: 51 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Laguna Negra and Human/HRP/02-72/BRA/2002

The SEQ ID NO: 52 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Maporal hantavirus.

The SEQ ID NO: 53 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantavirus Muju.

The SEQ ID NO: 54 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: New York, Sin

Nombre and Limestone Canyon.

The SEQ ID NO: 55 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Oran hantavirus. The SEQ ID NO: 56 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Prospect Hill, Topografov and Tula.

The SEQ ID NO: 57 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the Sangassou hantavirus.

The SEQ ID NO: 58 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Thailand 741 hantavirus.

The SEQ ID NO: 59 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Asama hantavirus.

The SEQ ID NO: 60 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Cao hantavirus.

The SEQ ID NO: 61 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the hantaviruses: Fusong-Mf-682,

Gou and CGRn9415.

The SEQ ID NO: 62 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the CGRn8316 hantavirus.

The SEQ ID NO: 63 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Z10 hantavirus.

The SEQ ID NO: 64 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and SEQ ID NO: 9 of Puumala virus and is also found in the Hokkaido hantavirus.

The SEQ ID NO: 65 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Jurong TJK/06(RT49) hantavirus. The SEQ ID NO: 66 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Montano hantavirus. The SEQ ID NO: 67 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Monongahela-2 hantavirus.

The SEQ ID NO: 68 is equivalent to the reference sequences SEQ ID NO: 4 of Andes virus and

SEQ ID NO: 9 of Puumala virus and is also found in the Oxbow hantavirus.

The SEQ ID NO: 69 is identical to the reference sequence SEQ ID NO: 5 of Andes virus and is also found in the hantaviruses: Araraquara Black Creek Canal, Laguna Negra,

Lechiguanas, New York, Oran, Rio Mamore, Sin Nombre, Montano and Monongahela-2. The SEQ ID NO: 70 is equivalent to the reference sequences SEQ ID NO: 5 of Andes virus and SEQ ID NO: 10 of Puumala virus and is also found in the Hokkaido hantavirus.

The SEQ ID NO: 71 is equivalent to the reference sequences SEQ ID NO: 5 of Andes virus and SEQ ID NO: 10 of Puumala virus and is also found in the Castelo dos Sonhos hantavirus.

The SEQ ID NO: 72 is equivalent to the reference sequences SEQ ID NO: 5 of Andes virus and SEQ ID NO: 10 of Puumala virus and is also found in the hantaviruses: Human/HRP/02-72/BRA/2002 and Limestone Canyon.

The SEQ ID NO: 73 corresponds a fragment of the nucleoprotein of Andes virus.

The SEQ ID NO: 74 corresponds to the Gc protein of Andes virus.

The SEQ ID NO: 75 corresponds to the Gc protein of Puumala virus. EXAMPLES

In these examples, the Andes virus (ANDV) from the Hantavirus genus is used to demonstrate that Gc fragments from the Hantavirus genus inhibit the fusion process and cell infection by hantaviruses. It is shown that Gc fragments do not produce a cytopathic effect in vitro and can be used to inhibit the infection of cells by the Andes hantavirus, specifically by blocking the membrane fusion process, thereby keeping Gc in a trimeric, unstable conformation. It is also shown that the fragments have an inhibitory effect on the Puumala virus, which is phylogenetically far from ANDV.

Example 1 : Synthesis of ANDV Gc fragments

Peptides comprising fragments from domain III and the predicted stem region of

ANDV Gc were prepared in a synthetic form. The peptides comprising fragments from ANDV Gc domain III, termed DIII-IA1 and DIII-IA2, encompass sequences EGAWGSGVGFTLT (SEQ ID NO: 1) and ARGSNTVKVVGK (SEQ ID NO: 2), respectively. Peptide R2, which comprises the C-terminal part of the stem region, encompasses sequence TFKCWFTKSGEWLLGILNGN (SEQ ID NO: 3). Peptide R2 was also divided into two even shorter peptides, R2.1 and R2.2, encompassing sequences TFKCWFTKSG (SEQ ID NO: 4) and EWLLGILNGN (SEQ ID NO: 5), respectively. As a negative control, peptide NN was used according to sequence SEQ ID NO: 73, derived from the ANDV nucleoprotein. The localization of the various peptides within the Gc sequence is shown in Fig. 1.

Example 2: In vitro cytotoxicity study of ANDV Gc fragments in cells.

A cellular proliferation essay was used to analyze the cytotoxicity of Gc fragments as detailed by the manufacturer (CellTiter®96, Promega). Briefly, the peptides were incubated with Vero E6 cells (ATTC) during 18 h at 37°C. Later, the conversion of tetrazolium to formazan was assessed by measuring the absorption at 490 nm (OD ₄₉₀ ) in a microplate reader (Synergy 4, BioTek). No alteration in the cellular metabolism was found at any of the peptide concentrations analyzed (Fig. 2).

Example 3: Fragments derived from ANDV Gc inhibit the infection of cells by ANDV. The infection of Vero E6 cells by ANDV (MOI = 0.1) was quantified by flow cytometry as previously established by our laboratory. Briefly, cells were incubated with ANDV isolate CHI- 7913 during 1 h at 37°C in the presence and absence of peptides. Subsequently, cells were washed and the infection was allowed to proceed during 16 h. In order to inactivate the virus, cells were later fixed with paraformaldehyde at 4% (p/v) and permeabilized with Triton X- 100 at 0.1% (v/v). For the indirect immunofluorescence analysis, cells were incubated during 45 min with a monoclonal anti-ANDV nucleoprotein antibody clone 7B3/F6. Subsequently, the primary antibody was detected with mouse anti- immunoglobulin conjugated to Alexa 555 (Life Technologies). More than 10,000 cells of each condition were analyzed using flow cytometry (FACS CAN II, Becton Dickinson). The standard deviation of at least 3 experiments is indicated as error bar for each value (Fig.3).

Among peptides derived from domain III, peptide DIII-IA2 was found to inhibit infection by around 50% at a concentration of 10-20 μΜ, while peptide DIII-IA1 showed lower inhibition (Fig. 3). On the other hand, peptide R2, which encompasses the C-terminal of the stem region, blocked cell infection by up to 55%, at a concentration of 20 uM (Fig. 3). By contrast, control peptide NN did not show any reduction in ANDV infection (Fig. 3).

Example 4: Fragments derived from Gc inhibit the formation of syncytia mediated by hantavirus glycoproteins.

In order to measure the fusogenic activity of the ANDV Gc protein, we used a cell fusion assay as previously used in the experimental section of CL51057. This test is based on transfecting Vero E6 cells with a plasmid that encodes the Gn and Gc glycoproteins from Andes virus (p. l8/GPC) or from Puumala virus (pWRG/PUUV-M(s2), kindly provided by Dr. Jay Hooper from USAMRIID, Fort Detrick, USA), where said sequences have been defined as SEQ ID NO: 74 and SEQ ID NO: 75, respectively. 48 h after transfection, the glycoproteins are present on the cell surface, as is well-known in the field. Since upon the endocytic uptake of the virus the Gc fusion protein becomes activated upon the low pH in endosomes, this protein can be activated on the cell surface by lowering the pH of the medium. This is carried out incubatiing the cells in E-MEM medium with pH 5.5 during 5 min at 37°C. At this point, the fusion protein on the cell surface is activated and leads to the fusion of the cell with adjacent cells. In order to inhibit fusion, the fragments derived from Gc are included in the low-pH medium. After incubation at a low pH, the medium is replaced by a common culture medium and cells are then incubated for 4 additional hours so that fused cells can reorganize their nuclei. Once the cells are fixed with 4% paraformaldehyde and the nuclei (DAPI), cytoplasm (CMFDA), and ANDV Gc protein (anti-Gc, Alexa 555) are stained, the cells are analyzed through fluorescence microscopy to quantify the number of cells and nuclei. If the quantity of cells is lower than that of nuclei, then the fused cells internally accumulated multiple nuclei, which is expressed by a high fusion index.

The inhibitory activity of Gc fragments in the membrane fusion process mediated by ANDV Gc was analyzed in this cell fusion assay at a concentration of 20 μΜ. 40% and 50% inhibition of Gc fusogenic activity was observed for peptides Dili IA1 and R2 respectively, while peptides R2.1, R2.2, and Dili IA2 inhibited more than 70% (Fig. 4A). In comparison, the NN negative control peptide did not show any inhibitory effect (Fig. 4A), which coincides with the results obtained with ANDV infection, which was also not inhibited by this control peptide (Fig. 3).

When the peptides were tested for cross inhibition on the fusion activity induced by the glycoproteins of PUUV, peptides Dili IA1, Dili IA2, R2, and R2.2, derived from ANDV Gc, showed cross inhibition of around 50% and peptide R2.1 inhibited PUUV Gc by around 70% (Fig. 4B). Once again, the negative control peptide NN did not show any inhibitory effect (Fig. 4B). Example 5: Peptides derived from ANDV Gc inhibit the fusion of ANDV with the plasma membrane.

Having found that Gc fragments inhibit cell-cell fusion, we went on to test if the peptides act directly on the fusion process induced by ANDV during the cell entry. To this end, ANDV was induced to fuse with the plasma membrane of cells. To this end, Vero E6 cells were pre-chilled on ice during 10 min in the presence of 20 mM of NH4C1. ANDV adsorption (MOI= 0.2) was later performed by an 1 h incubation at 4°C. The cells were then washed with pre-chilled PBS and the fusion of the virus with the plasma membrane was then triggered by incubation during 5 min at 37°C in an acidic medium (E-MEM, 20 mM of sodium succinate, pH 5.5) in the presence and absence of peptides. Subsequently, the cells were washed and the infection was allowed to proceed during 16 h at 37 °C in the presence of 20 mM NH ₄ C1. Cell infection was analyzed as described in example 3.

Using this experimental design, it was found that in a dose-dependent way, peptides Dili IA2 and R2 inhibited cell infection by up to 90% and 95%, respectively, at a concentration of 20 uM (Fig. 5). This test indicates that peptides directly act on the ANDV membrane fusion process.

Example 6: ANDV Gc peptides impede the formation of a stable Gc post-fusion trimer.

The Gc multimerization state was analyzed by saccharose gradient as previously established (Acuna et al , 2015). To this end, ANDV VLPs containing the ANDV Gn and Gc glycoproteins were produced, as described in our Chilean patent application, CL01085-2011. These VLPs were treated for 30 min at pH 5.5 to allow for a conformational change in Gc on the envelope of the viral particle. During this step, the VLPs were incubated in the presence and absence of Gc peptides. Subsequently, the glycoproteins were extracted using Triton X-100 at 1 % (v/v) and separated on a saccharose gradient of 7-15% (p/v) through ultracentrifugation at 150,000 g for 16 h. Gc presence was analyzed in each fraction by western blot using the monoclonal anti-ANDV Gc antibody clone 2H4/F6. The primary antibody was later detected with mouse anti-immunoglobulin conjugated to horseradish peroxidase (Sigma) using a chemiluminescent substrate (Pierce). The molecular mass of each fraction was determined independently through Coomassie staining of a molecular marker (Gel filtration standard, Bio- Rad) that was added to the same sedimentation gradient. The marker's experimental molecular mass was subsequently graphed against the log of the theoretical molecular mass indicated on the panel over the western blot (Fig. 6A). When the ANDV VLPs were incubated at pH 7, Gc was found mainly in fractions 3-8, corresponding to Gc monomers and probably dimers (Fig. 6A). When the VLPs were incubated at pH 5.5, a new Gc population could be observed peaking in fractions 9-10, corresponding to Gc homotrimers. On the other hand, when peptide R2 was added at a 20 μΜ concentration to the medium adjusted to pH 5.5, and next incubated during 30 min with the VLPs, Gc was localized in fractions 9-10, as also occurred in the absence of R2 (Fig. 6A). This data indicates that R2 did not alter Gc trimerization.

To test if the Gc trimer that had formed in the low-pH media in the presence of the R2 peptide was highly stable, as can be expected for a post-fusion homotrimer of class II fusion proteins, we went on to analyze the resistance of the Gc trimer to trypsin. To this end, the VLPs that were incubated for 30 min in media adjusted either to pH 7.0 or 5.5 in presence or absence of peptides. Subsequently, the VLPs were then incubated with TCPK trypsin (Sigma) for an additional 30 min. Digestion was stopped by adding SDS loading buffer and heating to 95 °C for 10 min. Gc digestion was then analyzed by western blot, using the monoclonal anti-ANDV Gc antibody clone 2H4/F6 as described above. As expected, when the VLP samples were first incubated at pH 7, and next incubated with TCPK trypsin, the complete digestion of Gc was observed. In contrast, when VLPs were incubated at pH 5.5 and then faced to TCPK trypsin, Gc showed resistance to trypsin action (Fig. 6B). On the other hand, when the R2 peptide was introduced in the low-pH incubation step and then faced to TCPK trypsin for 5 or 30 min, Gc was partially degraded, similarly to what happened at pH 7 (Fig. 6B). From these results, it can be concluded that incubation with R2 prevented Gc from adopting a stable post-fusion conformation resistant to trypsin, probably by preventing the stem region from folding over the trimeric core formed by domains I and II.

Finally, based on the experimental evidence shown in the realization examples, we can say the following:

- Fragments of domain III and the stem region of ANDV Gc do not produce cytotoxicity in cells in vitro.

- Fragments of domain III and the stem region of ANDV Gc inhibit the infection of cells by the Andes virus in vitro.

- Fragments of domain III and the stem region of ANDV Gc inhibit cell fusion mediated by ANDV and Puumala glycoproteins, which shows their cross reactivity among different hantavirus species.

- Fragments of domain III and the stem region of ANDV Gc block the infection of cells by ANDV, acting directly on the membrane fusion mechanism.

- Fragments of domain III and the stem region of ANDV Gc arrest the Gc membrane fusion protein in an intermediate state of the fusion process, retaining Gc in a trimeric state that is highly unstable. This instability interferes with fusion of the virus-cell membranes, impeding thereby the infection of cells by ANDV.

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