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SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2. By replacing the VSV glycoprotein with the spikes (S) of SARS-CoV-2 and SARS-CoV, we generated two replication-competent recombinant viruses, rVSV-SARS-CoV-2 and rVSV-SARS-CoV. Using wild-type and human ACE2 (hACE2) knock-in mouse models, we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal (i.n.) and intramuscular (i.m.) routes. Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV, no obvious cross-neutralizing activity was observed in the immunized mice sera. In macaques, neutralizing antibody (NAb) titers induced by one i.n. dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m. dose. Thus, our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n. administration instead of the traditional i.m. immunization in human. Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV-2 in a route-independent manner, we generated a chimeric antigen by replacing the receptor binding domain (RBD) of SARS-CoV S with that from the SARS-CoV-2. rVSV expressing the chimera (rVSV-SARS-CoV/2-RBD) induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2, with a safe Th1-biased response. Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV. hACE2 mice receiving a single i.m. dose of either rVSV-SARS-CoV-2 or rVSV-SARS-CoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs. Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

The geographic range of Zika virus (ZIKV) has expanded from Asia to the Americas, leading to the 2015-2016 pandemic with enhanced neurovirulence. At present, ZIKV is continuously circulating in many Southeast Asian countries. Unfortunately, the persistent evolution of ZIKV in Southeast Asia and its influence on the biological characteristics of the virus remain incompletely understood. In this study, the in vitro and in vivo properties of a new ZIKV isolate obtained from Cambodia in 2019 (CAM/2019) were characterized and compared with those of the Cambodian strain (CAM/2010). Compared with CAM/2010, the CAM/2019 virus showed similar plaque morphology and growth curves in cell cultures and induced comparable viremia and organ viral loads profiles in both BALB/c and A129 (IFNAR1

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

SARS-CoV-2 infection causes a wide spectrum of clinical manifestations in humans, and olfactory dysfunction is one of the most predictive and common symptoms in COVID-19 patients. However, the underlying mechanism by which SARS-CoV-2 infection leads to olfactory disorders remains elusive. Herein, we demonstrate that intranasal inoculation with SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), not the olfactory bulb (OB), resulting in transient olfactory dysfunction in humanized ACE2 (hACE2) mice. The sustentacular cells and Bowman’s gland cells in the OE were identified as the major target cells of SARS-CoV-2 before invasion into olfactory sensory neurons (OSNs). Remarkably, SARS-CoV-2 infection triggers massive cell death and immune cell infiltration and directly impairs the uniformity of the OE structure. Combined transcriptomic and quantitative proteomic analyses revealed the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptor (OR) genes in the OE from the infected animals. Overall, our mouse model recapitulates olfactory dysfunction in COVID-19 patients and provides critical clues for understanding the physiological basis for extrapulmonary manifestations of COVID-19.

Infectious virus diseases, particularly coronavirus disease 2019, have posed a severe threat to public health, whereas the developed therapeutic and prophylactic strategies are seriously challenged by viral evolution and mutation. Therefore, broad-spectrum inhibitors of viruses are highly demanded. Herein, an unprecedented antiviral strategy is reported, targeting the viral glycan shields with hypervalent mannose-binding nanoparticles. The nanoparticles exhibit a unique double-punch mechanism, being capable of not only blocking the virus-receptor interaction but also inducing viral aggregation, thereby allowing for inhibiting the virus entry and facilitating the phagocytosis of viruses. The nanoparticles exhibit potent and broad-spectrum antiviral efficacy to multiple pseudoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its major variants (D614G, N501Y, N439K, Δ69-70, Delta, and Omicron; lentiviruses expressing only the spike proteins), as well as other vital viruses (human immunodeficiency virus 1 and Lassa virus), with apparent EC50 values around the 10

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242–244 deletion (242–244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242–244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.

The newly identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a global health emergency because of its rapid spread and high mortality. The molecular mechanism of interaction between host and viral genomic RNA is yet unclear. We demonstrate herein that SARS-CoV-2 genomic RNA, as well as the negative-sense RNA, is dynamically N6-methyladenosine (m6A)-modified in human and monkey cells. Combined RIP-seq and miCLIP analyses identified a total of 8 m6A sites at single-base resolution in the genome. Especially, epidemic strains with mutations at these identified m6A sites have emerged worldwide, and formed a unique cluster in the US as indicated by phylogenetic analysis. Further functional experiments showed that m6A methylation negatively regulates SARS-CoV-2 infection. SARS-CoV-2 infection also triggered a global increase in host m6A methylome, exhibiting altered localization and motifs of m6A methylation in mRNAs. Altogether, our results identify m6A as a dynamic epitranscriptomic mark mediating the virus–host interaction.

Homologous and heterologous booster with COVID-19 mRNA vaccines represent the most effective strategy to prevent the ongoing Omicron pandemic. The additional protection from these prototype SARS-CoV-2 S-targeting vaccine was attributed to the increased RBD-specific memory B cells with expanded potency and breadth. Herein, we show the safety and immunogenicity of heterologous boosting with the RBD-targeting mRNA vaccine AWcorna (also term ARCoV) in Chinese adults who have received two doses inactivated vaccine. The superiority over inactivated vaccine in neutralization antibodies, as well as the safety profile, support the use of AWcorna as heterologous booster in China.

Zika virus (ZIKV) suddenly evolved from a neglected arthropod-borne flavivirus into a pandemic pathogen during 2015-2016. A panel of amino acid mutations has been shown to be responsible for the enhanced neurovirulence and transmissibility of ZIKV. Recent studies have demonstrated that ZIKV genomic RNA is modified by host N6-methyladenosine (m

The association of Zika virus (ZIKV) infection with microcephaly has raised alarm worldwide. Their causal link has been confirmed in different animal models infected by ZIKV. However, the molecular mechanisms underlying ZIKV pathogenesis are far from clear. Hence, we performed global gene expression analysis of ZIKV-infected mouse brains to unveil the biological and molecular networks underpinning microcephaly. We found significant dysregulation of the sub-networks associated with brain development, immune response, cell death, microglial cell activation, and autophagy amongst others. We provided detailed analysis of the related complicated gene networks and the links between them. Additionally, we analyzed the signaling pathways that were likely to be involved. This report provides systemic insights into not only the pathogenesis, but also a path to the development of prophylactic and therapeutic strategies against ZIKV infection.

West Nile virus (WNV) is an important neurotropic flavivirus that is widely distributed globally. WNV strain XJ11129 was first isolated in Xinjiang, China, and its genetic and biological characteristics remain largely unknown. In this study, phylogenetic and sequence analyses revealed that XJ11129 belongs to lineage 1a and shares high genetic identity with the highly pathogenic strain NY99. Then, the full-length genomic cDNA of XJ11129 was amplified and assembled using a modified Gibson assembly (GA) method. The virus (named rXJ11129) was successfully rescued in days following this method. Compared with other wild-type WNV isolates, rXJ11129 exhibited virulence indistinguishable from that of the NY99 strain in vivo. In summary, the genomic and virulence phenotypes of rXJ11129 were characterized in vivo and in vitro, and these data will improve the understanding of the spread and pathogenesis of this reemerging virus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00246-x) contains supplementary material, which is available to authorized users.

Modeling SARS-CoV-2 in mice Among the research tools necessary to develop medical interventions to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, high on the list are informative animal models with which to study viral pathogenesis. Gu et al. developed a mouse model in which a SARS-CoV-2 strain was infectious and could cause an inflammatory response and moderate pneumonia. Adaptation of this viral strain in the mouse appeared to be dependent on a critical amino acid change, Asn501 to Tyr (N501Y), within the receptor-binding domain of the viral spike protein. The new mouse model was used to study neutralizing antibodies and a vaccine candidate against the virus. Science, this issue p. 1603, A SARS-CoV-2 mouse model is used to study viral responses and the development of vaccine candidates., The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.

Zika virus (ZIKV) is an emerging mosquito-borne pathogen belonging to the genus Flavivirus of the family Flaviviridae. Aedes albopictus is widely distributed in China. However, little is known about the vector competence of Ae. albopictus in China. The present study presents the oral susceptibility and vector competence of Ae. albopictus Guangzhou strain to ZIKV. Additionally, vertical transmission of ZIKV is described. The results demonstrated the susceptibility of local Ae. albopictus mosquitoes to ZIKV with an extrinsic incubation period of 6 days. Disseminated infection was observed in Ae. albopictus starting on day 2 postinfection (PI). Starting on day 6 PI, the saliva of Ae. albopictus exhibited ZIKV infection, and the transmission rate was 36.4%. Vertical transmission was observed during the first gonotrophic cycle. The minimum infection rate was observed in third-to-fourth instar larvae.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are closely related flaviviruses, ZIKV circulates in the population that has been JEV vaccinated in Southeast Asian countries. This alerts that a pre-existing immunity to JEV would impact ZIKV infection and/or pathogenesis. Herein we showed that the pre-existing immunity to JEV SA14-14-2 vaccination provided an ample protection against non-lethal or lethal dose of ZIKV infection in mice. This was in sharp contrast to the passive immunization of JEV antibodies, which failed to affect ZIKV infection or pathogenesis in mice, albeit these antibodies exhibited cross-reactivity and antibody dependent enhancement (ADE) of ZIKV infection in vitro. Furthermore, we determined that JEV vaccine-elicited CD8+ T cells were required to mediate the heterotypic protection of ZIKV infection, which cross-reacted to ZIKV E and NS5 antigens (E294-302 and NS52839-2848). Adoptive transfer of these CD8+ T cells could partially protect the mice from ZIKV challenge. Therefore, although short of epidemiological evidence, these results suggested that cross-reactive CD8+ T cells activated by JEV vaccination could protect potential ZIKV infection in human populations.

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 15 (95% CI: 9.8–21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 350–375), which was Comparable to that of conventional qRT-PCR (740 copies/ml, 95% CI: 690–750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320–0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523–0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

Flaviviruses have posed a serious threat to human health in the past decades, and effective therapeutic drugs are lacking; thus, treatment of flavivirus infection is a great challenge. The flavivirus protease NS2B3 is an attractive target for antiviral drug screening. Here, we developed an intracellular Zika virus (ZIKV) NS2AB3 self-cleavage assay to identify inhibitors that interfere with viral polyprotein cleavage and block ZIKV/dengue virus (DENV) replication. Bortezomib was identified as the most potent inhibitor, with a half-maximal effective concentration (EC50) in the nanomolar range. We found that instead of directly inhibiting NS2B3 protease activity, bortezomib dramatically induced the ubiquitination and aggregation of NS3, leading to the attenuation of its protease activity in cells. Two E3 ligases, HRD1 and RNF126, were found to be responsible for NS3 ubiquitination. Our study identifies bortezomib as a potential drug for the treatment of ZIKV/DENV infection and reveals the central role of the ERAD pathway in the inhibition of flaviviruses by bortezomib.

Cellular and humoral immunity are both important in host defense against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although defects in SARS-CoV-2-specific T cell immunity have been found in patients with severe lung pathology, it is still largely unclear whether virus-specific T cells are sufficient for host protection. Here, we found that in a previously characterized cohort of convalescent subjects, one individual, though lacking detectable anti-viral neutralizing IgG antibodies, showed virus-specific T cell responses, both in CD4+ and CD8+ T cells. SARS-CoV-2-specific T cells in this and other individuals are maintained for up to 10 months. This study thus further supports a critical role of T cells in host defense against SARS-CoV-2, offering new insights into the design and evaluation of COVID-19 vaccines.

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine.One sentence summaryA sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

Significance Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a severe threat to public health and the global economy. Its spike protein is responsible for the membrane fusion and is thus a major target for vaccine and drug development. Our study presents the in situ structure of the spike protein in the postfusion state with higher resolution, giving further insights into the design of a viral entry inhibitor. Our observation of the oligomerization states of spikes on the viral membrane implies a possible mechanism of membrane fusion for viral infection., The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in β-propiolactone–inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N

Yellow fever virus (YFV) is a re-emerging flavivirus, which can lead to severe clinical manifestations and high mortality, with no specific antiviral therapies available. The live-attenuated yellow fever vaccine 17D (YF17D) has been widely used for over eighty years. However, the emergence of yellow fever vaccine-associated viscerotropic disease (YFL-AVD) and yellow fever vaccine-associated neurotropic disease (YFL-AND) raised non-negligible concerns. Additionally, the attenuation mechanism of YF17D is still unclear. Thus, the development of convenient models is crucial to understand the mechanisms behind YF17D attenuation and its adverse effects. In this work, we generated a reporter YF17D expressing nano-luciferase (NLuc). In vitro and in vivo characterization demonstrated that the NLuc-YF17D shared similar biological properties with its parental strain and the NLuc activity can reflect viral infectivity reliably. Combined with in vivo bioluminescence imaging, a series of mice models of YF17D infection was established, which will be useful for the evaluation of antiviral medicines and novel vaccine candidates. Especially, we demonstrated that intraperitoneally (i.p.) infection of NLuc-YF17D in type I interferon receptor-deficient mice A129 resulted in outcomes resembling YEL-AVD and YEL-AND, evidenced by viral replication in multiple organs and invasion of the central neuronal system. Finally, in vitro and in vivo assays based on this reporter virus were established to evaluate the antiviral activities of validated antiviral agents. In conclusion, the bioluminescent reporter virus described herein provides a powerful platform to study YF17D attenuation and vaccine-associated diseases as well as to develop novel countermeasures against YFV.

The circulation of Zika virus (ZIKV) in nearly 80 countries and territories poses a significant global threat to public health. ZIKV is causally linked to severe developmental defects in the brain, recognized as congenital Zika syndrome (CZS), which includes microcephaly and other serious congenital neurological complications. Since the World Health Organization declared the ZIKV outbreak a public health emergency of international concern, remarkable progress has been made in the generation of different ZIKV infection animal models to gain insight into cellular targets and pathogenesis and to explore the associated underlying mechanisms. Here we focus on summarizing our current understanding of the effects of ZIKV on mammalian brain development in different developmental stages and discuss the potential underlying mechanisms of ZIKV-induced CZS, as well as future perspectives.

Zika virus is a positive single-strand RNA virus whose replication involved RNA unwinding and synthesis. ZIKV NS3 contains a helicase domain, but its enzymatic activity is not fully characterized. Here, we established a dsRNA unwinding assay based on the FRET effect to study the helicase activity of ZIKV NS3, which provided kinetic information in real time. We found that ZIKV NS3 specifically unwound dsRNA/dsDNA with a 3′ overhang in the 3′ to 5′ direction. The RNA unwinding ability of NS3 significantly decreased when the duplex was longer than 18 base pairs. The helicase activity of NS3 depends on ATP hydrolysis and binding to RNA. Mutations in the ATP binding region or the RNA binding region of NS3 impair its helicase activity, thus blocking viral replication in the cell. Furthermore, we showed that ZIKV NS5 interacted with NS3 and stimulated its helicase activity. Disrupting NS3-NS5 interaction resulted in a defect in viral replication, revealing the tight coupling of RNA unwinding and synthesis. We suggest that NS3 helicase activity is stimulated by NS5; thus, viral replication can be carried out efficiently. Our work provides a molecular mechanism of ZIKV NS3 unwinding and novel insights into ZIKV replication.

The re-emergence of Zika virus (ZIKV) in the Western Hemisphere has resulted in global public health crisis since 2015. ZIKV preferentially infects and targets human neural progenitor cells (hNPCs) and causes fetal microcephaly upon maternal infection. hNPCs not only play critical roles during fetal brain development, but also persist in adult brain throughout life. Yet the mechanism of innate antiviral immunity in hNPCs remains largely unknown. Here, we show that ZIKV infection triggers the abundant production of virus-derived small interfering RNAs in hNPCs, but not in the more differentiated progenies or somatic cells. Ablation of key RNAi machinery components significantly enhances ZIKV replication in hNPCs. Furthermore, enoxacin, a broad-spectrum antibiotic that is known as an RNAi enhancer, exerts potent anti-ZIKV activity in hNPCs and other RNAi-competent cells. Strikingly, enoxacin treatment completely prevents ZIKV infection and circumvents ZIKV-induced microcephalic phenotypes in brain organoid models that recapitulate human fetal brain development. Our findings highlight the physiological importance of RNAi-mediated antiviral immunity during the early stage of human brain development, uncovering a novel strategy to combat human congenital viral infections through enhancing RNAi.

Sexual transmission of Zika Virus (ZIKV) elevates the risk of its dissemination in the female reproductive tract and causes a serious threat to the fetus. However, the available animal models are not appropriate to investigate sexual transmission, dynamics of ZIKV infection, replication, and shedding. The use of tree shrew as a small animal model of ZIKV vaginal infection was assessed in this study. A total of 23 sexually mature female tree shrews were infected with ZIKV GZ01viathe intravaginal route. There was no significant difference in change of body weight, and the temperature between ZIKV infected and control animals. Viral RNA loads were detected in blood, saliva, urine, and vaginal douching. ZIKV RNA was readily detected in vaginal lavage of 22 animals (95.65%, 22/23) at 1 dpi, and viral load ranged from 104.46 to 107.35 copies/ml, and the peak of viral load appeared at 1 dpi. The expression of key inflammatory genes, such as IL6, 8, CCL5, TNF-a, and CXCL9, was increased in the spleen of ZIKV infected animals. In the current study, female tree shrews have been successfully infected with ZIKV through the vaginal route for the first time. Interestingly, at first, ZIKV replicates at the local site of infection and then spreads throughout the host body to develop a robust systemic infection and mounted a protective immune response. This small animal model is not only valuable for exploring ZIKV sexual transmission and may also help to explain the cause of debilitating manifestations of the fetusin vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces new-onset diabetes and severe metabolic complications of pre-existing diabetes. The pathogenic mechanism underlying this is incompletely understood. Here, we provided evidence linking circulating GP73 with the exaggerated gluconeogenesis triggered by SARS-CoV-2 infection. We found that SARS-CoV-2 infection or glucotoxic conditions increased GP73 production and secretion. Secreted GP73 then trafficked to the liver and kidney to stimulate gluconeogenesis through the cAMP/PKA pathway. By using global phosphoproteomics, we found a drastic remodeling of the PKA kinase hub exerted by GP73. Notably, plasma GP73 levels were elevated and positively correlated with blood glucose in patients with COVID19 and diabetes. Neutralization of circulating GP73 in serum of individuals infected with SARS-CoV-2 or with diabetes reduced excessive gluconeogenesis in cultured hepatocytes, and lowered blood glucose levels in animal models of diabetes. Ablation of GP73 from whole animals has a profound glucose-lowering effect secondary to reduced gluconeogenesis. Thus, GP73 is a key glucogenic hormone contributing to SARS-CoV-2-induced glucose abnormality.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces new-onset diabetes and severe metabolic complications of pre-existing diabetes. The pathogenic mechanism underlying this is incompletely understood. Here, we provided evidence linking circulating GP73 with the exaggerated gluconeogenesis triggered by SARS-CoV-2 infection. We found that SARS-CoV-2 infection or glucotoxic condition increased the cellular secretion of GP73. Secreted GP73 trafficked to the liver and kidney to stimulate gluconeogenesis through cAMP/PKA pathway. By using global phosphoproteomics, we found a drastic remodeling of PKA kinase hub exerted by GP73. Notably, COVID-19 patients showed pathologically elevated plasma GP73, and neutralization of the secreted GP73 inhibited enhanced PKA signaling and glucose production associated with SARS-CoV-2 infection. GP73 blockade also reduced gluconeogenesis and lowered hyperglycemia in type 2 (T2D) diabetic mice. Therefore, our findings provide novel insight into the roles of GP73 as a key glucogenic hormone and mechanistic clues underlying the development of SARS-CoV-induced glucose abnormalities.

Comprehensive profiling of humoral antibody response to severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) proteins is essential in understanding the host immunity and in developing diagnostic tests and vaccines. To address this concern, we developed a SARS-CoV-2 proteome peptide microarray to analyze antibody interactions at the amino acid resolution. With the array, we demonstrate the feasibility of employing SARS-CoV-1 antibodies to detect the SARS-CoV-2 nucleocapsid phosphoprotein. The first landscape of B-cell epitopes for SARS-CoV-2 IgM and IgG antibodies in the serum of 10 coronavirus disease of 2019 (COVID-19) patients with early infection is also constructed. With array data and structural analysis, a peptide epitope for neutralizing antibodies within the SARS-CoV-2 spike receptor-binding domain's interaction interface with the angiotensin-converting enzyme 2 receptor was predicted. All the results demonstrate the utility of our microarray as a platform to determine the changes of antibody responses in COVID-19 patients and animal models as well as to identify potential targets for diagnosis and treatment.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has resulted in ~1,119,431 deaths. There is currently no approved vaccines or therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein promotes entry into host cells and is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target the SARS-CoV-2 Spike protein. One human antibody, CA521LALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521LALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. The LALA mutation introduced to CA521 abrogates the binding with Fc receptors or complement receptors reducing antibody-dependent enhancement seen with anti-SARS-CoV antibodies. CA521LALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521LALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with the virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521LALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 receptor binding domain (RBD) in the Spike protein. CA521LALA blocks the interaction by binding all three RBDs of one SARS CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions in the treatment of COVID-19 and identifies CA521LALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10 times the effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to the ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the receptor binding domain, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.

The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain, named MASCp36, that causes severe acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was 58 PFU in 9-month-old, male BALB/c mice. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three mutations in RBD significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of human ACE2 (hACE2) or mACE2 in complex with the RBD of MASCp36 at 3.1 to 3.7 angstrom resolution elucidates molecular basis for the receptor-binding switch driven by specific amino acid substitutions. Interestingly, N501Y and Q493H enhanced the binding affinity to human ACE2 (hACE2); while triple mutations N501Y/Q493H/K417N decreased affinity to hACE2, thus led to the reduced infectivity of MASCp36 to human cells. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in human and animals.One sentence summaryA mouse adapted SARS-CoV-2 strain that harbored specific amino acid substitutions in the RBD of S protein showed 100% mortality in aged, male BALB/c mice.

SummaryReceptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10-fold of effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the RBD, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.HighlightsSARS-CoV-2 specific antibody, HB27, blocks viral receptor binding and membrane fusionHB27 confers prophylactic and therapeutic protection against SARS-CoV-2 in mice modelsRhesus macaques showed no adverse side effects when administered with HB27Cryo-EM studies suggest that HB27 sterically occludes SARS-CoV-2 from its receptor

Olfactory dysfunction caused by SARS-CoV-2 infection represents as one of the most predictive and common symptoms in COVID-19 patients. However, the causal link between SARS-CoV-2 infection and olfactory disorders remains lacking. Herein we demonstrate intranasal inoculation of SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), resulting in transient olfactory dysfunction in humanized ACE2 mice. The sustentacular cells and Bowman’s gland cells in OE were identified as the major targets of SARS-CoV-2 before the invasion into olfactory sensory neurons. Remarkably, SARS-CoV-2 infection triggers cell death and immune cell infiltration, and impairs the uniformity of OE structure. Combined transcriptomic and proteomic analyses reveal the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptors in OE from the infected animals. Overall, our mouse model recapitulates the olfactory dysfunction in COVID-19 patients, and provides critical clues to understand the physiological basis for extrapulmonary manifestations of COVID-19.

Author(s): Zu, Shulong; Deng, Yong-Qiang; Zhou, Chao; Li, Jie; Li, Lili; Chen, Qi; Li, Xiao-Feng; Zhao, Hui; Gold, Sarah; He, Jun; Li, Xiang; Zhang, Changqing; Yang, Heng; Cheng, Genhong; Qin, Cheng-Feng

Summary Influenza A virus (IAV) infection stimulates a type I interferon (IFN-I) response in host cells that exerts antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). However, most ISGs are poorly studied for their roles in the infection of IAV. Herein, we demonstrate that SERTA domain containing 3 (SERTAD3) has a significant inhibitory effect on IAV replication in vitro. More importantly, Sertad3−/− mice develop more severe symptoms upon IAV infection. Mechanistically, we find SERTAD3 reduces IAV replication through interacting with viral polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acidic protein (PA) to disrupt the formation of the RNA-dependent RNA polymerase (RdRp) complex. We further identify an 8-amino-acid peptide of SERTAD3 as a minimum interacting motif that can disrupt RdRp complex formation and inhibit IAV replication. Thus, our studies not only identify SERTAD3 as an antiviral ISG, but also provide the mechanism of potential application of SERTAD3-derived peptide in suppressing influenza replication.

Replication-competent vesicular stomatitis virus (VSV)-based recombinant viruses are useful tools for studying emerging and highly pathogenic enveloped viruses in level 2 biosafety facilities. Here, we used a replication-competent recombinant VSVs (rVSVs) encoding the spike (S) protein of SARS-CoV-2 in place of the original G glycoprotein (rVSV-eGFP-SARS-CoV-2) to develop a high-throughput entry assay for SARS-CoV-2. The S protein was incorporated into the recovered rVSV-eGFP-SARS-CoV-2 particles, which could be neutralized by sera from convalescent COVID-19 patients. The recombinant SARS-CoV-2 also displayed entry characteristics similar to the wild type virus, such as cell tropism and pH-dependence. The neutralizing titers of antibodies and sera measured by rVSV-eGFP-SARS-CoV-2 were highly correlated with those measured by wild-type viruses or pseudoviruses. Therefore, this is a safe and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines and therapeutics.

SUMMARYThe World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, the basis of which remains largely unclear. Currently, though convalescent individuals have been shown with both cellular and humoral immune responses, there is very limited understanding on the immune responses, especially adaptive immune responses, in patients with severe COVID-19. Here, we examined 10 blood samples from COVID-19 patients with acute respiratory distress syndrome (ARDS). The majority of them (70%) mounted SARS-CoV-2-specific humoral immunity with production of neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, accompanied with decreased IFNγ expression in CD4+ T cells in peripheral blood from severe patients. Most notably, we failed in detecting SARS-CoV-2-specific IFNγ production by peripheral blood lymphocytes from these patients. Our work thus indicates that COVID-19 patients with severe symptoms are associated with defective cellular immunity, which not only provides insights on understanding the pathogenesis of COVID-19, but also has implications in developing an effective vaccine to SARS-CoV-2.

Background The clinical management of coronavirus disease 2019 (COVID-19) is dependent on understanding the underlying factors that contribute to the disease severity. In the absence of effective antiviral therapies, other host immunomodulatory therapies such as targeting inflammatory response are currently being used without clear evidence of their effectiveness. Because inflammation is an essential component of host antiviral mechanisms, therapies targeting inflammation may adversely affect viral clearance and disease outcome. Objective To understand whether the persistent presence of the virus is a key determinant in the disease severity during COVID-19 and to determine whether the viral reactivation in some patients is associated with infectious viral particles. Methods The data for patients were available including the onset of the disease, duration of viral persistence, measurements of inflammatory markers such as IL-6 and C-reactive protein, chest imaging, disease symptoms, and their durations among others. Follow-up tests were performed to determine whether the viral negative status persists after their recovery. Results Our data show that patients with persistent viral presence (>16 days) have more severe disease outcomes including extensive lung involvement and requirement of respiratory support. Two patients who died of COVID-19 were virus-positive at the time of their death. Four patients demonstrated virus-positive status on the follow-up tests, and these patient samples were sent to viral culture facility where virus culture could not be established. Conclusions These data suggest that viral persistence is the key determining factor of the disease severity. Therapies that may impair the viral clearance may impair the host recovery from COVID-19.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese encephalitis (JE), the most commonly diagnosed viral encephalitis worldwide. The fatality rate of JE is 20%, and nearly half of the surviving patients develop neuropsychiatric sequelae. Axl is a receptor tyrosine kinase that plays multiple roles in flaviviral infections. Currently, the involvement of Axl in JEV infection remains enigmatic. In this study, we demonstrate that Axl impedes the pathogenesis of severe JE in mice by maintaining blood-brain-barrier (BBB) integrity and restricting viral neuroinvasion. Furthermore, serum IL-1α is a key mediator of this process and is primarily released by JEV-infected pyroptotic macrophages to elicit BBB breakdown, while an IL-1α antagonist can effectively reduce the incidence of severe JE. Our work uncovers the protective role of Axl in antagonizing severe JE and shows that the use of an IL-1α antagonist may be a promising tactic to prevent severe JE., Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis (JE), which has an unclear pathogenesis. Despite vaccination, thousands of deaths attributed to JE are reported annually. In this study, we report that mice deficient for Axl, a receptor tyrosine kinase that plays multiple roles in flaviviral infection, displayed greater mortality upon JEV infection. The effect of Axl deficiency on JEV infection was mediated by markedly elevated serum interleukin-1α (IL-1α) levels, which devastated the blood-brain-barrier and promoted viral neuroinvasion within 24 h postinfection. Using an in situ infection model, we showed that dead macrophages were the primary source of observed increased serum IL-1α levels. Axl deficiency enhanced cell death and caused pyroptosis in 80% of JEV-infected macrophages by disrupting phosphatidylinositol 3-kinase (PI3K)-Akt signaling. Intriguingly, the primary effector released by pyroptotic macrophages in our model was IL-1α rather than IL-1β. Finally, we assessed the effect of an IL-1α antagonist and demonstrated that it effectively prevented the incidence of JE. Our results indicate that Axl plays a protective role in JEV infection, identify IL-1α released by pyroptotic macrophages as a crucial factor promoting JEV neuroinvasion, and suggest that an IL-1α antagonist may be a candidate for JE therapy. IMPORTANCE Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes Japanese encephalitis (JE), the most commonly diagnosed viral encephalitis worldwide. The fatality rate of JE is 20%, and nearly half of the surviving patients develop neuropsychiatric sequelae. Axl is a receptor tyrosine kinase that plays multiple roles in flaviviral infections. Currently, the involvement of Axl in JEV infection remains enigmatic. In this study, we demonstrate that Axl impedes the pathogenesis of severe JE in mice by maintaining blood-brain-barrier (BBB) integrity and restricting viral neuroinvasion. Furthermore, serum IL-1α is a key mediator of this process and is primarily released by JEV-infected pyroptotic macrophages to elicit BBB breakdown, while an IL-1α antagonist can effectively reduce the incidence of severe JE. Our work uncovers the protective role of Axl in antagonizing severe JE and shows that the use of an IL-1α antagonist may be a promising tactic to prevent severe JE.