From Viral Entry to Genome Replication: Mapping the Druggable Weak Points of Hantaviruses

Orthohantaviruses, rodent-borne enveloped viruses with tri-segmented single-stranded negative-sense RNA genomes, remain among the most dangerous yet therapeutically neglected human pathogens. They cause two life-threatening clinical syndromes: hemorrhagic fever with renal syndrome (HFRS) across Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas, the latter carrying case fatality rates that can approach or exceed 50 percent. Despite decades of research and recurring outbreaks, including a highly publicized Andes virus outbreak aboard a cruise ship in April 2026, there is still no universally licensed antiviral or vaccine against orthohantavirus infection. A recent review published in Frontiers in Cellular and Infection Microbiology by researchers at the University of São Paulo and Institut Pasteur de São Paulo systematically maps the viral life cycle for chemotherapeutic intervention, evaluating entry inhibitors, polymerase blockers, nucleocapsid disruptors, and host-directed strategies while advocating for computational approaches to accelerate discovery.

The authors frame their analysis around the fundamental steps of the orthohantavirus infection cycle. Viral entry begins when the Gn glycoprotein docks onto host surface receptors, including β3-integrins and protocadherin-1 (PCDH1). Once internalized via endocytosis, the acidification of the endosomal compartment triggers a conformational change in the viral envelope that drives membrane fusion, releasing the viral genome into the cytoplasm. The viral RNA-dependent RNA polymerase (RdRp) then initiates both transcription and genome replication. Structural proteins and newly synthesized RNA genomes assemble at the Golgi complex or plasma membrane before budding out as mature infectious virions. Each of these stages presents a distinct molecular vulnerability that chemists and virologists can exploit with rationally designed small molecules, peptides, or biologics.

Among the most extensively characterized entry inhibitors are cyclic peptides identified through phage display libraries. Early work identified cyclic nonapeptides that bind the β3-integrin receptor used by pathogenic orthohantaviruses such as Sin Nombre virus (SNV), Hantaan virus (HTNV), Seoul virus (SEOV), and Puumala virus (PUUV) to enter human endothelial cells. Of seventy sequences screened, eleven showed inhibitory effects exceeding 80 percent against SNV in vitro, with the most potent, CPFVKTQLC, demonstrating high inhibitory potency when presented on phage particles. This peptide was subsequently refined through alanine substitution and deletion scanning to a minimal functional unit, the cyclic pentapeptide cyclo-[CPFVC]. However, because free monovalent peptides proved less effective than phage-displayed versions, researchers pivoted toward multivalent presentation on nanoparticles, targeting the viral glycoproteins directly rather than the host receptor. When the peptide CLVRNLAWC was displayed on 100-nanometer nanoparticles at a 4:1 ratio, inhibition of SNV rose from 9 percent as a free peptide to 32.5 percent. The peptide CQATTARNC achieved even greater efficacy, surpassing 50 percent inhibition when the nanoparticle-to-virus ratio was increased to 20:1.

Building on these strategies, investigators extended the approach to Andes virus (ANDV) by eluting peptide libraries from purified virions using monoclonal antibodies against the Gn and Gc glycoproteins. A key finding was that combining peptides targeting different Gn- and Gc-specific sequences produced synergistic inhibition significantly more powerful than single-peptide treatments, underscoring the value of simultaneously engaging multiple viral epitopes. To transition from biological peptides to drug-like molecules, researchers used the three-dimensional structure of the refined pentapeptide cyclo-[CPFVC] in a ligand-based virtual screening campaign evaluating over 600,000 small molecules. This effort identified compounds such as G319–0078 and 8012-0652, which were approximately 2,000 times more potent than the original cyclic peptides, achieving high-affinity inhibition in the nanomolar range with EC50 values between 1.3 and 2.5 nanomolar in vitro. These molecules represent a compelling proof of concept that computational screening can transform a modest peptide lead into a highly potent preclinical candidate warranting further in vivo evaluation.

A distinct strategy targets the membrane fusion process itself. The orthohantavirus Gc glycoprotein functions as a class II fusion protein, and completing membrane fusion requires a major structural relocation of Domain III (DIII) to achieve the post-fusion state. Exploiting this mechanical vulnerability, researchers demonstrated that viral entry can be blocked using recombinant fragments and synthetic peptides derived from the Gc DIII and stem region. These fragments allow initial Gc trimerization to proceed but ultimately prevent membrane hemifusion and fusion pore formation. The recombinant DIII fragment inhibited ANDV fusion by approximately 70 percent at 25 micromolar and demonstrated cross-reactivity by reducing PUUV-mediated fusion by 50 percent. Even greater potency was observed with the R2 peptide, derived from the C-terminal portion of the Gc stem region, which inhibited ANDV fusion by up to 95 percent in vitro. Although efficacy dropped to a 70 percent cross-reduction against PUUV-mediated fusion at 20 micromolar due to high genetic divergence between the two viruses, the results still demonstrate meaningful cross-protective potential.

Natural products have also yielded promising entry inhibitors. Griffithsin (GRFT), an algae-derived lectin with demonstrated efficacy against coronaviruses, retroviruses, lentiviruses, flaviviruses, hepaciviruses, paramyxoviruses, and herpesviruses, potently inhibits ANDV entry in vitro. A synthetic trimeric variant possessing nine oligosaccharide-binding sites showed even greater potency against ANDV and SNV. In vivo, GRFT inhibited HTNV and provided partial protection in suckling mice. Its scalability and ease of purification highlight its translational potential. Separately, bovine lactoferrin (LF) prevents initial viral adsorption and adhesion of SEOV, likely by interacting with cellular glycosaminoglycans such as heparin or by sterically hindering virus binding to host β-integrin and lectin receptors. Prophylactic administration of LF one day before orthohantavirus inoculation improved survival rates in mice up to 70 percent at 160 milligrams per kilogram, with a double-administration regimen enhancing survival to over 94 percent. When combined with subsequent ribavirin treatment, the two agents functioned synergistically, profoundly reducing viral shedding and completely inhibiting viral protein expression.

Discovered through high-throughput screening of over 2,300 approved drugs and traditional Chinese medicines, cepharanthine (CEP), a compound isolated from Stephania cephalantha Hayata, has emerged as a potent viral entry and membrane fusion inhibitor. In vitro, CEP inhibited HTNV pseudovirus with an EC50 of 0.9 micromolar and reduced viral nucleic acid copies by 73 percent at 20 micromolar. In vivo, administration to BALB/c mice five hours post-infection significantly reduced infection by 93 percent at 90 milligrams per kilogram per day and 94 percent at 180 milligrams per kilogram per day. Given its established safety profile, CEP represents a high-priority candidate for repurposing and advancement into human clinical trials against orthohantaviruses.

On the replication front, ribavirin remains the historical benchmark for anti-hantaviral therapy. Once transported into host cells, ribavirin is phosphorylated into metabolites including ribavirin-5′-monophosphate, which inhibits inosine monophosphate dehydrogenase and depletes intracellular GTP, and ribavirin-5′-triphosphate, which selectively interacts with the viral RdRp as its primary mechanism of action. Incorporation of ribavirin leads to accumulation of genomic errors and dramatic loss of virus viability through error catastrophe. Preclinical evaluation confirmed that early ribavirin administration reduces viral loads and prevents severe disease in suckling mouse models infected with HTNV and hamster models infected with ANDV. Clinically, intravenous ribavirin demonstrated efficacy in a double-blind, placebo-controlled trial of 242 patients with HTNV-induced HFRS, significantly reducing hemorrhagic and overall mortality. However, efficacy varies significantly across strains and clinical syndromes. In the context of HCPS caused by ANDV, ribavirin intervention eliminated mortality compared to a 35 percent historical case-fatality rate and reduced the incidence of the severe cardiopulmonary phase from 84 to 57 percent. Conversely, in a larger placebo-controlled, double-blinded trial evaluating intravenous ribavirin for HCPS, the drug failed to demonstrate overall therapeutic efficacy, revealing no significant difference in survival between groups. Trials in HFRS caused by PUUV also showed insufficient efficacy and safety, likely due to delayed drug administration and a weaker correlation between viral load and disease severity compared to HTNV infection. Furthermore, the clinical utility of ribavirin is constrained by dose-limiting toxicities, most notably reversible hemolytic anemia reported in 71 percent of recipients.

Favipiravir (T-705), originally discovered during a screening for anti-influenza compounds, has emerged as a potent viral replication inhibitor. Upon cellular entry, host enzymes convert favipiravir into its active form, favipiravir-RTP, a purine nucleotide analogue incorporated into nascent viral RNA that induces lethal mutagenesis. Favipiravir potently inhibited ANDV and SNV in vitro and provided 100 percent survival in lethal ANDV hamster models when treatment was initiated before viremia, making it an important candidate for post-exposure prophylaxis. Combination with ribavirin produced highly synergistic effects, with peak antiviral activity observed at a ratio of 12 micromolar favipiravir to 18.75 micromolar ribavirin, achieving greater than 99 percent overall inhibition of HTNV. Additionally, ETAR (1-β-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole) demonstrated high potency in vitro against HTNV and ANDV, and in vivo studies using HTNV-challenged suckling mice showed approximately 25 percent protection at doses of just 12.5 and 25 milligrams per kilogram, mirroring ribavirin’s therapeutic outcomes at a fraction of the concentration. Beyond nucleoside analogues, the viral nucleoprotein has emerged as a promising alternative target. Terpene-isoindole conjugates such as compound 18 demonstrated potent anti-HTNV activity with exceptionally low toxicity, and molecular docking suggests they bind to the highly conserved RNA-binding site of the nucleoprotein, competing with viral RNA and effectively arresting intracellular replication. Similarly, morin, a natural flavonoid found in plants like mulberry and perilla, binds directly to the core assembly region of the viral nucleoprotein, sterically hindering RNA encapsidation and disrupting the formation of functional replication complexes.

In the absence of licensed antiviral treatments, clinical management remains primarily supportive, focusing on intensive monitoring and treatment of respiratory, cardiac, and renal complications. Achieving early diagnosis remains a primary clinical challenge, as the efficacy of supportive care is substantially reduced if initiated after the onset of severe disease. Host-targeted and anti-inflammatory strategies have recently been explored to mitigate the characteristic vascular leak and cytokine storm, most notably during the 2026 cruise ship outbreak, where the JAK inhibitor baricitinib and the bradykinin B2 receptor antagonist icatibant were administered in a combined therapeutic regimen alongside ribavirin and favipiravir. Passive immunotherapy continues to be a major investigative focus, with convalescent plasma showing potential to reduce mortality and newer candidates such as the quadrivalent human polyclonal antibody SAB-163 and highly potent monoclonal antibodies demonstrating up to 100 percent protection in lethal animal models. Regarding prevention, Hantavax has been used to prevent HFRS in South Korea, and China routinely administers inactivated bivalent vaccines targeting HTNV and SEOV in endemic regions. However, a globally licensed orthohantavirus vaccine does not currently exist, leaving New World strains and HCPS without an approved preventative countermeasure. The review’s authors argue that expanding preclinical testing to include globally neglected variants, combined with modern in silico screening of extensive chemical and natural product libraries, will be essential to develop broad-spectrum hantaviral therapeutics capable of mitigating future epidemic threats.

Subject of Research: Antiviral drug discovery targeting the orthohantavirus life cycle

Article Title: Hantaviral therapeutics: life cycle targets and chemotherapeutic interventions

Article References: Kocabiyik, D. Z., de Araujo, J., Durigon, E. L., & Wrenger, C. (2026). Hantaviral therapeutics: life cycle targets and chemotherapeutic interventions. Frontiers in Cellular and Infection Microbiology, 16, Article 1972830. https://doi.org/10.3389/fcimb.2026.1972830

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1972830

Keywords: orthohantavirus, antiviral discovery, viral entry inhibitors, RNA-dependent RNA polymerase, ribavirin, favipiravir, griffithsin, cepharanthine, nucleoprotein inhibitors, HFRS, HCPS, virtual screening