Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Effectiveness of COVID-19 vaccines administered in the 2023 autumnal campaigns in Europe: Results from the VEBIS primary care test-negative design study, September 2023-January 2024.

In autumn 2023, European vaccination campaigns predominantly administered XBB.1.5 vaccine. In a European multicentre study, we estimated 2023 COVID-19 vaccine effectiveness (VE) against laboratory-confirmed symptomatic infection at primary care level between September 2023 and January 2024. Using a test-negative case-control design, we estimated VE in the target group for COVID-19 vaccination overall and by time since vaccination. We included 1057 cases and 4397 controls. Vaccine effectiveness was 40 % (95 % CI: 26-53 %) overall, 48 % (95 % CI: 31-61 %) among those vaccinated < 6 weeks of onset and 29 % (95 % CI: 3-49 %) at 6-14 weeks. Our results suggest that COVID-19 vaccines administered to target groups during the autumn 2023 campaigns showed clinically significant effectiveness against laboratory-confirmed, medically attended symptomatic SARS-CoV-2 infection in the 3 months following vaccination. A longer study period will allow for further variant-specific COVID-19 VE estimates, better understanding decline in VE and informing booster administration policies.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Hardening of Respiratory Syncytial Virus Inclusion Bodies by Cyclopamine Proceeds through Perturbation of the Interactions of the M2-1 Protein with RNA and the P Protein.

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.

Spatial resolution of virus replication: RSV and cytoplasmic inclusion bodies.

Respiratory Syncytial Virus (RSV) is a major cause of respiratory illness in young children, elderly and immunocompromised individuals worldwide representing a severe burden for health systems. The urgent development of vaccines or specific antivirals against RSV is impaired by the lack of knowledge regarding its replication mechanisms. RSV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Mononegavirales order (MNV) which includes other viruses pathogenic to humans as Rabies (RabV), Ebola (EBOV), or measles (MeV) viruses. Transcription and replication of viral genomes occur within cytoplasmatic virus-induced spherical inclusions, commonly referred as inclusion bodies (IBs). Recently IBs were shown to exhibit properties of membrane-less organelles (MLO) arising by liquid-liquid phase separation (LLPS). Compartmentalization of viral RNA synthesis steps in viral-induced MLO is indeed a common feature of MNV. Strikingly these key compartments still remain mysterious. Most of our current knowledge on IBs relies on the use of fluorescence microscopy. The ability to fluorescently label IBs in cells has been key to uncover their dynamics and nature. The generation of recombinant viruses expressing a fluorescently-labeled viral protein and the immunolabeling or the expression of viral fusion proteins known to be recruited in IBs are some of the tools used to visualize IBs in infected cells. In this chapter, microscope techniques and the most relevant studies that have shed light on RSV IBs fundamental aspects, including biogenesis, organization and dynamics are being discussed and brought to light with the investigations carried out on other MNV.

Ribonucleoprotein transport in Negative Strand RNA viruses.

Negative-sense, single-stranded RNA (-ssRNA) viruses comprise some of the deadliest human pathogens (Ebola, rabies, influenza A viruses etc.). Developing therapeutic tools relies on a better understanding of their multiplication cycle. For these viruses, the genome replication and transcription activities most-often segregate in membrane-less environments called inclusion bodies (IBs) or viral factories. These "organelles" usually locate far from the cell surface from where new virions are released, and -ssRNA viruses do not encode for transport factors. The efficient trafficking of the genome progeny toward the cell surface is most often ensured by mechanisms co-opting the cellular machineries. In this review, for each -ssRNA viral family, we cover the methods employed to characterize these host-virus interactions, the strategies used by the viruses to promote the virus genome transport, and the current gaps in the literature. Finally, we highlight how Rab11 has emerged as a target of choice for the intracellular transport of -ssRNA virus genomes.

Respiratory Syncytial Virus Two-Step Infection Screen Reveals Inhibitors of Early and Late Life Cycle Stages.

Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available. We aimed to discover new and chemically diverse candidates to enrich the hRSV drug development pipeline. We used a two-step screen that interrogates compound efficacy after primary infection and a consecutive virus passaging. We resynthesized selected hit molecules and profiled their activities with hRSV lentiviral pseudotype cell entry, replicon, and time-of-addition assays. The breadth of antiviral activity was tested against recent RSV clinical strains and human coronavirus (hCoV-229E), and in pseudotype-based entry assays with non-RSV viruses. Screening 6,048 molecules, we identified 23 primary candidates, of which 13 preferentially scored in the first and 10 in the second rounds of infection, respectively. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses, including hCoV-229E. One compound, identified in the second round of infection, did not measurably inhibit hRSV cell entry or replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated BCi-NS1.1 lung cells. In conclusion, we identified four new hRSV inhibitor candidates with different modes of action. Our findings build an interesting platform for medicinal chemistry-guided derivatization approaches followed by deeper phenotypical characterization in vitro and in vivo with the aim of developing highly potent hRSV drugs.

Retinoic Acid-Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection.

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.

Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking.

Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

A condensate-hardening drug blocks RSV replication in vivo.

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.

Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection.

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Performance of 30 commercial SARS-CoV-2 serology assays in testing symptomatic COVID-19 patients.

We report evaluation of 30 assays' (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation.

Molecular investigation of a RSV outbreak in a geriatric hospital.

BACKGROUND: Acquired infections in hospitalized elderly people are a growing concern. In long-term care facilities with multiple staff and visitor contacts, virus outbreaks are a common challenge for infection prevention teams. Although several studies have reported nosocomial RSV outbreaks in long term care facilities, molecular epidemiology data are scarce. METHODS: RSV RNA was detected in respiratory samples from 19 patients in a long-term care hospital for elderly in Paris in March 2019 over a 3 weeks period. Genotyping was performed using nucleotide sequencing. Sociodemographic and clinical characteristics of cases part of a unique cluster, were retrospectively reviewed. RESULTS: Molecular investigation of theses RSV cases, revealed a unique cluster of 12 nosocomial cases in 2 adjacent wards. Mean age of these outbreak's cases was 89. All patients had underlying medical conditions. Seven exhibited lower respiratory symptoms and three experienced decompensation of underlying chronic heart condition. Two patients died. CONCLUSIONS: This case report highlights the importance of RSV in causing substantial disease in elderly in case of nosocomial outbreak and the contributions of molecular epidemiology in investigation and management of such outbreak.

New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution.

BACKGROUND: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus-host interactions and related acute respiratory disease, at the level of the entire lung. METHODS: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. RESULTS: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. CONCLUSIONS: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.

Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON's antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs.

Clinical Cutaneous Features of Patients Infected With SARS-CoV-2 Hospitalized for Pneumonia: A Cross-sectional Study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a current pandemic worldwide. This virus can reach all organs and disturbs the immune system, leading to a cytokine storm in severe forms. We aimed to report cutaneous features among coronavirus disease 2019 (COVID-19) hospitalized patients. METHODS: We performed a cross-sectional study on 1 given day among all patients hospitalized in acute care for COVID-19 and included all patients with cutaneous features. Follow-up 48 hours later was obtained. RESULTS: Among 59 adult patients hospitalized on the day of the study in an infectious diseases ward for SARS-CoV-2 infection who were confirmed by molecular assay and/or radiological findings (computed tomography scan), 40 were included. Several cutaneous manifestations were found: macular exanthema (80%), face edema (32%), livedo (13%), urticarial rash (8%), purpura (5%), oral lichenoid lesions (33%), and conjunctivitis (18%). Cutaneous biopsy was performed in 17 patients. Histological findings showed mast cell hyperplasia (100%), superficial perivascular infiltrate of lymphocytes (94%), and superficial edema (47%) consistent with capillary leak. CONCLUSIONS: Various dermatological signs can be encountered during COVID-19. A macular rash was the most frequent. All cutaneous features could be related to a vascular leak process.

Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro.

Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics.IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.