Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.

Egg-adaptive mutations of human influenza H3N2 virus are contingent on natural evolution.

Egg-adaptive mutations in influenza hemagglutinin (HA) often emerge during the production of egg-based seasonal influenza vaccines, which contribute to the largest share in the global influenza vaccine market. While some egg-adaptive mutations have minimal impact on the HA antigenicity (e.g. G186V), others can alter it (e.g. L194P). Here, we show that the preference of egg-adaptive mutation in human H3N2 HA is strain-dependent. In particular, Thr160 and Asn190, which are found in many recent H3N2 strains, restrict the emergence of L194P but not G186V. Our results further suggest that natural amino acid variants at other HA residues also play a role in determining the preference of egg-adaptive mutation. Consistently, recent human H3N2 strains from different clades acquire different mutations during egg passaging. Overall, these results demonstrate that natural mutations in human H3N2 HA can influence the preference of egg-adaptation mutation, which has important implications in seed strain selection for egg-based influenza vaccine.

Multi-cohort analysis of depression-associated gut bacteria sheds insight on bacterial biomarkers across populations.

Gut microbes are associated with the development of depression based on extensive evidence. However, previous studies have led to conflicting reports on this association, posing challenges to the application of gut bacteria in the diagnostics and treatment of depression. To minimise heterogenicity in data analysis, the present meta-analysis adopted a standardised bioinformatics and statistical pipeline to analyse 16S rRNA sequences of 1827 samples from eight different cohorts. Although changes in the overall bacterial community were identified by our meta-analysis, depressive-correlated changes in alpha-diversity were absent. Enrichment of Bacteroidetes, Parabacteroides, Barnesiella, Bacteroides, and Bacteroides vulgatus, along with depletion in Firmicutes, Dialister, Oscillospiraceae UCG 003 and UCG 002, and Bacteroides plebeius, were observed in depressive-associated bacteria. By contrast, elevated L-glutamine degradation, and reduced L-glutamate and L-isoleucine biosynthesis were identified in depressive-associated microbiomes. After systemically reviewing the data of these collected cohorts, we have established a bacterial classifier to identify depressive symptoms with AUC 0.834 and 0.685 in the training and external validation dataset, respectively. Moreover, a low-risk bacterial cluster for depressive symptoms was identified, which was represented by a lower abundance of Escherichia-Shigella, and a higher abundance of Faecalibacterium, Oscillospiraceae UCG 002, Ruminococcus, and Christensenellaceae R.7 group.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling.

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12(-/-) Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12(-/-) cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.

Fc receptors in antibody-dependent enhancement of viral infections.

Sensitization of the humoral immune response to invading viruses and production of antiviral antibodies forms part of the host antiviral repertoire. Paradoxically, for a number of viral pathogens, under certain conditions, antibodies provide an attractive means of enhanced virus entry and replication in a number of cell types. Known as antibody-dependent enhancement (ADE) of infection, the phenomenon occurs when virus-antibody immunocomplexes interact with cells bearing complement or Fc receptors, promoting internalization of the virus and increasing infection. Frequently associated with exacerbation of viral disease, ADE of infection presents a major obstacle to the prevention of viral disease by vaccination and is thought to be partly responsible for the adverse effects of novel antiviral therapeutics such as intravenous immunoglobulins. There is a growing body of work examining the intracellular signaling pathways and epitopes responsible for mediating ADE, with a view to aiding rational design of antiviral strategies. With in vitro studies also confirming ADE as a feature of infection for a growing number of viruses, challenges remain in understanding the multilayered molecular mechanisms of ADE and its effect on viral pathogenesis.

Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection.

To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.

CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus.

BACKGROUND: Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. METHODS: We have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens. RESULTS: We show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional information on the role of downstream signaling by FcγRII. CONCLUSIONS: These results demonstrate that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to FcγRII receptors.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses.

Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.

Corrigendum: XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants.

[This corrects the article DOI: 10.3389/fimmu.2021.761250.].

Age-related seroprevalence trajectories of seasonal coronaviruses in children including neonates in Guangzhou, China.

OBJECTIVES: Four seasonal coronaviruses, including human coronavirus (HCoV)-229E and HCoV-OC43, HCoV-NL63, and HCoV-HKU1 cause approximately 15-30% of common colds in adults. However, the full landscape of the immune trajectory to these viruses that covers the whole childhood period is still not well understood. METHODS: We evaluated the serological responses against the four seasonal coronaviruses in 1886 children aged under 18 years by using enzyme-linked immunosorbent assay. The optical density values against each HCoV were determined from each sample. Generalized additive models were constructed to determine the relationship between age and seroprevalence throughout the whole childhood period. The specific antibody levels against the four seasonal coronaviruses were also tested from the plasma samples of 485 pairs of postpartum women and their newborn babies. RESULTS: The immunoglobulin (Ig) G levels of the four seasonal coronaviruses in the mother and the newborn babies were highly correlated (229E: r = 0.63; OC43: r = 0.65; NL63: r = 0.69; HKU1: r = 0.63). The seroprevalences in children showed a similar trajectory in that the levels of IgG in the neonates dropped significantly and reached the lowest level after the age of around 1 year (229E: 1.18 years; OC43: 0.97 years; NL63: 1.01 years; HKU1: 1.02 years) and then resurgence in the children who aged older than 1 year. Using the lowest level from the generalized additive models as our cutoff, the seroprevalences for HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 were 98.11%, 96.23%, 96.23% and 94.34% at the age of 16-18 years. CONCLUSION: Mothers share HCoV-specific IgGs with their newborn babies and the level of maternal IgGs waned at around 1 year after birth. The resurgence of the HCoV-specific IgGs was found thereafter with the increase in age suggesting repeated infection occurred in children.

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway.

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.

Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells.

Shigella flexneri, the causative agent of bacillar dystentery, invades the colonic mucosa where it elicits an intense inflammatory reaction responsible for destruction of the epithelium. During cell invasion, contact with host cells activates the type-III secretion of the Shigella IpaB and IpaC proteins. IpaB and IpaC are inserted into host cell plasma membranes and trigger initial signals that result in actin polymerization, while allowing cytosolic access of other bacterial effectors that further reorganize the cytoskeleton. After internalization, Shigella moves intracellularly and forms protrusions that infect neighbouring cells, promoting bacterial dissemination across the epithelium. Here, we show that during cell invasion, Shigella induces transient peaks in intracellular calcium concentration that are dependent on a functional type-III secretory apparatus. In addition, Shigella invasion induces the opening of Connexin 26 (Cx26) hemichannels in an actin- and phospholipase-C-dependent manner, allowing release of ATP into the medium. The released ATP, in turn, increases bacterial invasion and spreading, as well as calcium signalling induced by Shigella. These results provide evidence that pathogen-induced opening of connexin channels promotes signalling events that favour bacterial invasion and dissemination.

In vivo evidence for the involvement of the carboxy terminal domain in assembling connexin 36 at the electrical synapse.

Connexin 36 (Cx36)-containing electrical synapses contribute to the timing and amplitude of neural responses in many brain regions. A Cx36-EGFP transgenic was previously generated to facilitate their identification and study. In this study we demonstrate that electrical coupling is normal in transgenic mice expressing Cx36 from the genomic locus and suggest that fluorescent puncta present in brain tissue represent distributed electrical synapses. These qualities emphasize the usefulness of the Cx36-EGFP reporter as a tool for the detailed anatomical characterization of electrical synapses in fixed and living tissue. However, though the fusion protein is able to form gap junctions between Xenopus laevis oocytes it is unable to restore electrical coupling to interneurons in the Cx36-deficient mouse. Further experiments in transgenic tissue and non-neural cell lines reveal impaired transport to the plasma membrane as the possible cause. By analyzing the functional deficits exhibited by the fusion protein in vivo and in vitro, we identify a motif within Cx36 that may interact with other trafficking or scaffold proteins and thereby be responsible for its incorporation into electrical synapses.

XAV-19, a Swine Glyco-Humanized Polyclonal Antibody Against SARS-CoV-2 Spike Receptor-Binding Domain, Targets Multiple Epitopes and Broadly Neutralizes Variants.

Amino acid substitutions and deletions in the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBMs), in direct contact sites with the angiotensin converting enzyme-2 (ACE-2). Therefore, in Spike/ACE-2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study on Vero E6 cells conducted over 1 month, no mutation was associated with the addition of increasing doses of XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE-2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (COVID-19) including the different variants of concern identified so far.

Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles.

Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn-/- cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations - one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.