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SARS-CoV-2 has evolved into a panel of variants of concern (VOCs) and constituted a sustained threat to global health. The wildtype (WT) SARS-CoV-2 isolates fail to infect mice, while the Beta variant, one of the VOCs, has acquired the capability to infect standard laboratory mice, raising a spreading risk of SARS-CoV-2 from humans to mice. However, the infectivity and pathogenicity of other VOCs in mice remain not fully understood. In this study, we systematically investigated the infectivity and pathogenicity of three VOCs, Alpha, Beta, and Delta, in mice in comparison with two well-understood SARS-CoV-2 mouse-adapted strains, MASCp6 and MASCp36, sharing key mutations in the receptor-binding domain (RBD) with Alpha or Beta, respectively. Our results showed that the Beta variant had the strongest infectivity and pathogenicity among the three VOCs, while the Delta variant only caused limited replication and mild pathogenic changes in the mouse lung, which is much weaker than what the Alpha variant did. Meanwhile, Alpha showed comparable infectivity in lungs in comparison with MASCp6, and Beta only showed slightly lower infectivity in lungs when compared with MASCp36. These results indicated that all three VOCs have acquired the capability to infect mice, highlighting the ongoing spillover risk of SARS-CoV-2 from humans to mice during the continued evolution of SARS-CoV-2, and that the key amino acid mutations in the RBD of mouse-adapted strains may be referenced as an early-warning indicator for predicting the spillover risk of newly emerging SARS-CoV-2 variants.

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.

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

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

Summary There has been an urgent need of vaccines against coronavirus disease 2019 (COVID-19) due to the ongoing SARS-CoV-2 pandemic. Among all approaches, messenger RNA (mRNA) -based vaccine has emerged as a rapid and versatile platform to quickly respond to such a challenge. Here, we developed a lipid-nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (termed ARCoV). Intramuscular immunization of ARCoV mRNA-LNPs elicited robust neutralizing antibodies against SARS-CoV-2 as well as Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse adapted strain. Additionally, ARCoV was manufactured in liquid formulation and can be stored at room temperature for at least one week. This novel COVID-19 mRNA vaccine, ARCoV, is currently being evaluated in phase 1 clinical trials., Highlights Development of LNP-encapsulated mRNA vaccine (ARCoV) targeting the RBD of SARS-CoV-2 ARCoV induces neutralizing antibodies and T-cell immunity in mice and NHPs. ARCoV vaccination confers full protection against SARS-CoV-2 challenge in mice. ARCoV is a thermostable vaccine candidate for phase I studies, ARCoV is an LNP-encapsulated mRNA vaccine platform that is highly immunogenic and safe in mice and non-human primates, conferring protection against the challenge a SARS-CoV-2 mouse adapted strain.

Summary Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for animal model. Human Angiotensin converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) using CRISPR/Cas9 knock-in technology. Compared with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected- aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was evidenced to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis, and evaluating COVID-19 vaccines and therapeutics., Graphical Abstract, Highlights ● Human ACE2 knock-in mice were generated using CRISPR/Cas9 technology ● SARS-CoV-2 leads to robust replication in lung, trachea and brain ● SARS-CoV-2 causes interstitial pneumonia and elevated cytokine in aged hACE2 mice ● High dose of SARS-CoV-2 can establish infection via intragastric route in hACE2 mice, The COVID-19 pandemic has brought an urgent need for small animal models. Here, Sun et al. established an ACE2 humanized mouse by CRISPR/Cas9 knock-in technology. This hACE2 mice are susceptible to SARS-CoV-2 infection upon intranasal inoculation, and the resulting pulmonary infection and pathological changes resemble those observed in COVID-19 patients.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation. IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5ʹ–3ʹ exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3ʹ-UTR. The 3ʹ-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Zika virus (ZIKV) is a single-stranded, positive-sense RNA virus that is mainly transmitted by mosquito biting. ZIKV belongs to the family Flaviviridae , genus Flavivirus , which includes other important human pathogens including dengue virus, yellow fever virus, West Nile virus, and Japanese encephalitis virus. ZIKV was first isolated from a sentinel rhesus monkey in the Zika forest of Uganda in 1947. Following the outbreak of ZIKV in Yap Island of Micronesia in 2007 and the largest Zika pandemic in Brazil in 2015, ZIKV has rapidly spread through the Pacific islands and the Americas, resulting in an estimated two million infections per year. Due to its unexpected causal link to microcephaly in fetus and Guillain-Barre syndrome in adults, the current ongoing ZIKV epidemic was declared as a public health emergency of international concern by the World Health Organization in February, 2016. Accumulated evidence has demonstrated that ZIKV preferentially targets human neuronal progenitor cells, disrupts proliferation and differentiation, and triggers a strong immune response, resulting in significant neuronal cell death and microcephaly phenotype. Currently, no vaccines or specific antiviral drugs are available to prevent or treat ZIKV infections. Upon ZIKV infection, host cells immediately initiate several lines of antiviral innate immune responses, including the production of type I interferon (IFN) and its downstream signaling pathways. Type I IFN response is well evidenced to play a critical role during ZIKV infection, and mice deficient in type I IFN receptor are highly susceptible to ZIKV infection upon various injection routes. Type I IFN and its downstream signaling molecules (e.g. IFITM1 and IFITM3) show significant anti-ZIKV effects in both cell culture and animal models. Meanwhile, ZIKV has evolved diverse strategies to antagonize the host defense system through blocking the production of type I IFN and its downstream signaling, or escaping from antiviral effects of individual ISGs. Like other mosquito-borne flaviviruses, the viral proteins NS2B, NS3, NS4B and NS5, as well as the subgenomic flavivirus RNA (sfRNA), have been identified as potent suppressors of type I IFN induction and its effector pathways. Especially, ZIKV NS5 directly binds to STAT2, and leads to the proteasomal degradation of STAT2, resulting in the silencing of type I IFN signaling. These remarkable achievements in recent two years not only lead to the better understanding of ZIKV infection and pathogenesis, but also provide useful clues for the development of antiviral drugs and vaccine candidates. Several ISGs and their enzymatic products, including 25-Hydroxycholesterol, have been well characterized with potent anti-ZIKV activity. A few recombinant live-attenuated ZIKV vaccine candidates have been rationally constructed via reverse genetics with increased IFN sensitivity, which attributed to the attenuation phenotype in immunocompetent animals. Here, we summarize recent advances in the interplay between ZIKV and host type I IFN-mediated immune response.

Zika virus (ZIKV) has evolved from an overlooked mosquito-borne flavivirus into a global health threat due to its astonishing causal link to microcephaly and other disorders. ZIKV has been shown to infect neuronal progenitor cells of the fetal mouse brain, which is comparable to the first-trimester human fetal brain, and result in microcephaly. However, whether there are different effects between the contemporary ZIKV strain and its ancestral strain in the neonatal mouse brain, which is comparable with the second-trimester human fetal brain, is unclear. Here we adopted a mouse model which enables us to study the postnatal effect of ZIKV infection. We show that even 100 pfu of ZIKV can replicate and infect neurons and oligodendrocytes in most parts of the brain. Compared with the ancestral strain from Cambodia (CAM/2010), infection of the ZIKV strain from Venezuela (VEN/2016) leads to much more severe microcephaly, accompanied by more neuronal cell death, abolishment of oligodendrocyte development, and a more dramatic immune response. The serious brain damage caused by VEN/2016 infection would be helpful to elucidate why the American strain resulted in severe neurovirulence in infants and will provide clinical guidance for the diagnosis and treatment of infection by different ZIKV strains., Highlights • The infection of an American strain of ZIKV leads to more severe microcephaly than the ancestral Asian strain. • American strain infects more cells, and induces more dramatic immune response and cell death than ancestral Asian strain. World attention has been drawn to a global Zika virus (ZIKV) outbreak due to its unexpected causal link to congenital brain abnormalities, especially microcephaly. Infection of pregnant women with the American Zika strain, but not the ancestral Asian strain, can result in microcephaly in infants. However, the phenotypic difference between the contemporary American strain and ancestral Asian strain of ZIKV is still unclear. We employed the ZIKV infection model of a neonatal mouse brain to compare the difference between these two strains. We find that infection by the American strain leads to more severe microcephaly than the ancestral Asian strain.

The recent Zika virus (ZIKV) epidemic in Latin America coincided with a marked increase in microcephaly in newborns. However, the causal link between maternal ZIKV infection and malformation of the fetal brain has not been firmly established. Here we show a vertical transmission of ZIKV in mice and a marked effect on fetal brain development. We found that intraperitoneal (i.p.) injection of a contemporary ZIKV strain in pregnant mice led to the infection of radial glia cells (RGs) of dorsal ventricular zone of the fetuses, the primary neural progenitors responsible for cortex development, and caused a marked reduction of these cortex founder cells in the fetuses. Interestingly, the infected fetal mice exhibited a reduced cavity of lateral ventricles and a discernable decrease in surface areas of the cortex. This study thus supports the conclusion that vertically transmitted ZIKV affects fetal brain development and provides a valuable animal model for the evaluation of potential therapeutic or preventative strategies.

The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development., Given the recent Zika virus (ZIKV) epidemic, development of an effective vaccine is of high importance. Here, the authors use a licensed live-attenuated flavivirus vaccine backbone to develop a ZIKV vaccine and determine immunogenicity, safety and protection profiles in different animal models.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis -acting replication elements (5′-SLA and 5′-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.

Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection invitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role ofCH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.

Mutation for microcephaly Zika virus infections in humans have been known since 1947. Microcephaly and neuropathologies associated with Zika have only been reported recently, most prevalently in the Americas. Yuan et al. investigated recent stable mutations in the virus genome and engineered them into a low-virulence ancestral strain (see the Perspective by Screaton and Mongkolsapaya). A single amino acid substitution (serine to asparagine, S139N) in the viral precursor membrane protein exacerbated symptoms in pregnant mice. The reverse mutation (N139S) was less virulent. The S139N mutation arose in 2013 in French Polynesia before the virus jumped to Brazil in 2015. In vitro, this amino acid change made the virus more infectious for mouse and human neural progenitor cells and promoted apoptosis. The terrible sequelae of infection during pregnancy could thus be the result of a simple viral mutation. Science , this issue p. 933 ; see also p. 863

Zika virus (ZIKV) has raised global public health concerns due to its rapid transmission and unexpected clinical manifestations.1 No vaccine or antiviral treatment is currently available, and the d...

Flavivirus includes a large group of human pathogens with medical importance. Especially, neurotropic flaviviruses capable of invading central and peripheral nervous system, e.g. Japanese encephalitis virus (JEV) and Zika virus (ZIKV), are highly pathogenic to human and constitute major global health problems. However, the dynamic dissemination and pathogenesis of neurotropic flavivirus infections remain largely unknown. Here, using JEV as a model, we rationally designed and constructed a recombinant reporter virus that stably expressed Renilla luciferase (Rluc). The resulting JEV reporter virus (named Rluc-JEV) and parental JEV exhibited similar replication and infection characteristics, and the magnitude of Rluc activity correlated well with progeny viral production in vitro and in vivo. By using in vivo bioluminescence imaging (BLI) technology, we dissected the replication and dissemination dynamics of JEV infection in mice upon different inoculation routes. Interestingly, besides replicating in mouse brain, Rluc-JEV predominantly invaded the abdominal organs in mice with typical viscerotropism. Further tests in mice deficient in type I interferon (IFN) receptors demonstrated robust and prolonged viral replication in the intestine, spleen, liver, kidney and other abdominal organs. Combined with histopathological and immunohistochemical results, the host type I IFN signaling was evidenced as the major barrier to the viscerotropism and pathogenicity of this neurotropic flavivirus. Additionally, the Rluc-JEV platform was readily adapted for efficacy assay of known antiviral compounds and a live JE vaccine. Collectively, our study revealed abdominal organs as important targets of JEV infection in mice and profiled the unique viscerotropism trait controlled by the host type I IFN signaling. This in vivo visualization technology described here provides a powerful tool for testing antiviral agents and vaccine candidates for flaviviral infection.

Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus. IMPORTANCE Mosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile, and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus, DTMUV (also known as duck egg drop syndrome flavivirus [DEDSV]) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other nonavian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.