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Leaching of per- and polyfluoroalkyl substances (PFAS) during the post-consumer disposal of food contact materials (FCMs) poses a potential environmental threat but has seldom been evaluated. This study characterized the leaching behavior of PFAS and unidentified precursors from six common FCMs and assessed the impact of environmental conditions on PFAS release during disposal. The total concentration of 21 PFAS ranged from 3.2 to 377 ng/g in FCMs, with PFAS leachability into water varying between 1.1-42.8 %. Increasing temperature promoted PFAS leaching, with leached nine primary PFAS (∑9PFAS) reaching 46.3, 70.4, and 102 ng/L at 35, 45, and 55 â, respectively. Thermodynamic analysis (∆G>0, ∆H>0, and ∆S<0) indicated hydrophobic interactions control PFAS leaching. The presence of dissolved organic matter in synthetic leachate increased the leached ∑9PFAS from 47.1 to 103 ng/L but decreased PFBS, PFOS, and 6:2 FTS leaching. The total release of seven perfluorocarboxylic acids (∑7PFCAs) from takeaway food packaging waste was estimated to be 0.3-8.2 kg/y to landfill leachate and 0.6-15.4 kg/y to incineration plant leachate, contributing 0.2-4.8 % and 0.1-3.2 % of total ∑7PFCAs in each leachate type. While the study presents a refined methodology for estimating PFAS release during disposal, future research is needed on the indirect contribution from precursors.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22,995 and 28,866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , SARS-CoV-2/genética , Células CACO-2 , Recombinación Homóloga , Glicoproteína de la Espiga del CoronavirusRESUMEN
Increasing evidence shows the African lineage Zika virus (ZIKV) displays a more severe neurovirulence compared to the Asian ZIKV. However, viral determinants and the underlying mechanisms of enhanced virulence phenotype remain largely unknown. Herein, we identify a panel of amino acid substitutions that are unique to the African lineage of ZIKVs compared to the Asian lineage by phylogenetic analysis and sequence alignment. We then utilize reverse genetic technology to generate recombinant ZIKVs incorporating these lineage-specific substitutions based on an infectious cDNA clone of Asian ZIKV. Through in vitro characterization, we discover a mutant virus with a lysine to arginine substitution at position 101 of capsid (C) protein (termed K101R) displays a larger plaque phenotype, and replicates more efficiently in various cell lines. Moreover, K101R replicates more efficiently in mouse brains and induces stronger inflammatory responses than the wild type (WT) virus in neonatal mice. Finally, a combined analysis reveals the K101R substitution promotes the production of mature C protein without affecting its binding to viral RNA. Our study identifies the role of K101R substitution in the C protein in contributing to the enhanced virulent phenotype of the African lineage ZIKV, which expands our understanding of the complexity of ZIKV proteins.
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Infección por el Virus Zika , Virus Zika , Animales , Ratones , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Sustitución de Aminoácidos , Filogenia , Replicación Viral/genéticaRESUMEN
Zika virus (ZIKV) infection during pregnancy threatens pregnancy and fetal health. However, the infectivity and pathological effects of ZIKV on placental trophoblast progenitor cells in early human embryos remain largely unknown. Here, using human trophoblast stem cells (hTSCs), we demonstrated that hTSCs were permissive to ZIKV infection, and resistance to ZIKV increased with hTSC differentiation. Combining gene knockout and transcriptome analysis, we demonstrated that the intrinsic expression of AXL and TIM-1, and the absence of potent interferon (IFN)-stimulated genes (ISGs) and IFNs contributed to the high sensitivity of hTSCs to ZIKV. Furthermore, using our newly developed hTSC-derived trophoblast organoid (hTSC-organoid), we demonstrated that ZIKV infection disrupted the structure of mature hTSC-organoids and inhibited syncytialization. Single-cell RNA sequencing (scRNA-seq) further demonstrated that ZIKV infection of hTSC-organoids disrupted the stemness of hTSCs and the proliferation of cytotrophoblast cells (CTBs) and probably led to a preeclampsia (PE) phenotype. Overall, our results clearly demonstrate that hTSCs represent the major target cells of ZIKV, and a reduced syncytialization may result from ZIKV infection of early developing placenta. These findings deepen our understanding of the characteristics and consequences of ZIKV infection of hTSCs in early human embryos.
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Infección por el Virus Zika , Virus Zika , Embarazo , Humanos , Femenino , Trofoblastos , Placenta , OrganoidesRESUMEN
Virus spillover remains a major challenge to public health. A panel of SARS-CoV-2-related coronaviruses have been identified in pangolins, while the infectivity and pathogenicity of these pangolin-origin coronaviruses (pCoV) in humans remain largely unknown. Herein, we comprehensively characterized the infectivity and pathogenicity of a recent pCoV isolate (pCoV-GD01) in human cells and human tracheal epithelium organoids and established animal models in comparison with SARS-CoV-2. pCoV-GD01 showed similar infectivity to SARS-CoV-2 in human cells and organoids. Remarkably, intranasal inoculation of pCoV-GD01 caused severe lung pathological damage in hACE2 mice and could transmit among cocaged hamsters. Interestingly, in vitro neutralization assays and animal heterologous challenge experiments demonstrated that preexisting immunity induced by SARS-CoV-2 infection or vaccination was sufficient to provide at least partial cross-protection against pCoV-GD01 challenge. Our results provide direct evidence supporting pCoV-GD01 as a potential human pathogen and highlight the potential spillover risk.
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Municipal solid wastes (MSWs) contain diverse per- and polyfluoroalkyl substances (PFAS), and these substances may leach into leachates, resulting in potential threats to the environment and human health. In this study, leachates from incineration plants with on-site treatment systems were measured for 17 PFAS species, including 13 perfluorocarboxylic acids (PFCAs) and 4 perfluorosulfonic acids (PFSAs). PFAS were detected in all of the raw leachates and finished effluents in concentrations ranging from 7228 to 16,565 ng L-1 and 43 to 184 ng L-1, respectively, with a greater contribution from the short-chain PFAS and PFCAs. The results showed that the existing combined processes (biological treatment and membrane filtration) were effective in decreasing PFAS in the aqueous phase with removal efficiencies over 95%. In addition, correlation analysis suggested that physical entrapment, not biodegradation, was the main means of PFAS reduction in the treatment system. These results filled a gap in the understanding of PFAS occurrence and removal in leachates from incineration plants during the full-scale treatment processes, and demonstrated those leachates were previously under-explored sources of PFAS.
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Fluorocarburos , Contaminantes Químicos del Agua , Humanos , Incineración , Residuos Sólidos/análisis , Fluorocarburos/análisis , Contaminantes Químicos del Agua/análisisRESUMEN
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.
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Proteínas del Envoltorio Viral , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Modelos Animales de Enfermedad , Polisacáridos/química , Proteínas del Envoltorio Viral/genética , Virulencia , Replicación Viral/genética , Virus Zika/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
SARS-CoV-2 variants have posed significant challenges to the hopes of using ancestral strain-based vaccines to address the risk of breakthrough infection by variants. We designed and developed a bivalent vaccine based on SARS-CoV-2 Alpha and Beta variants (named SCTV01C). SCTV01C antigens were stable at 25 oC for at least 6 months. In the presence of a squalene-based oil-in-water adjuvant SCT-VA02B, SCTV01C showed significant protection efficacy against antigen-matched Beta variant, with favorable safety profiles in rodents. Notably, SCTV01C exhibited cross-neutralization capacity against Omicron subvariants (BA.1, BA.1.1, BA.2, BA.3, and BA.4/5) in mice, superior to a WT (D614G)-based vaccine, which reinforced our previously published findings that SCTV01C exhibited broad-spectrum neutralizing potencies against over a dozen pre-Omicron variants and the Omicron BA.1 variant. In summary, variant-based multivalent protein vaccine could be a platform approach to address the challenging issues of emerging variants, vaccine hesitancy and the needs of affordable and thermal stable vaccines.
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COVID-19 , Vacunas Virales , Ratones , Humanos , Animales , SARS-CoV-2/genética , Vacunas Combinadas , Vacunas Virales/genética , Escualeno , COVID-19/prevención & control , Anticuerpos Antivirales , Agua , Anticuerpos NeutralizantesRESUMEN
Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.
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Infección por el Virus Zika , Virus Zika , Animales , Cobayas , Humanos , Inmunidad Humoral , Transmisión Vertical de Enfermedad Infecciosa , Viremia , Virus Zika/fisiologíaRESUMEN
The yellow fever (YF) live attenuated vaccine strain 17D (termed 17D) has been widely used for the prevention and control of YF disease. However, 17D retains significant neurovirulence and viscerotropism in mice, which is probably linked to the increased occurrences of serious adverse events following 17D vaccination. Thus, the development of an updated version of the YF vaccine with an improved safety profile is of high priority. Here, we generated a viable bicistronic YF virus (YFV) by incorporating the internal ribosome entry site (IRES) from Encephalomyocarditis virus into an infectious clone of YFV 17D. The resulting recombinant virus, 17D-IRES, exhibited similar replication efficiency to its parental virus (17D) in mammalian cell lines, while it was highly restricted in mosquito cells. Serial passage of 17D-IRES in BHK-21 cells showed good genetic stability. More importantly, in comparison with the parental 17D, 17D-IRES displayed significantly decreased mouse neurovirulence and viscerotropism in type I interferon (IFN)-signaling-deficient and immunocompetent mouse models. Interestingly, 17D-IRES showed enhanced sensitivity to type I IFN compared with 17D. Moreover, immunization with 17D-IRES provided solid protection for mice against a lethal challenge with YFV. These preclinical data support further development of 17D-IRES as an updated version for the approved YF vaccine. This IRES-based attenuation strategy could be also applied to the design of live attenuated vaccines against other mosquito-borne flaviviruses. IMPORTANCE Yellow fever (YF) continually spreads and causes epidemics around the world, posing a great threat to human health. The YF live attenuated vaccine 17D is considered the most efficient vaccine available and helps to successfully control disease epidemics. However, side effects may occur after vaccination, such as viscerotropic disease (YEL-AVD) and neurotropic adverse disease (YEL-AND). Thus, there is an urgent need for a safer YF vaccine. Here, an IRES strategy was employed, and a bicistronic YFV was successfully developed (named 17D-IRES). 17D-IRES showed effective replication and genetic stability in vitro and high attenuation in vivo. Importantly, 17D-IRES induced humoral and cellular immune responses and conferred full protection against lethal YFV challenge. Our study provides data suggesting that 17D-IRES, with its prominent advantages, could be a vaccine candidate against YF. Moreover, this IRES-based bicistronic technology platform represents a promising strategy for developing other live attenuated vaccines against emerging viruses.
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Interferón Tipo I , Vacuna contra la Fiebre Amarilla , Fiebre Amarilla , Ratones , Humanos , Animales , Fiebre Amarilla/prevención & control , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Sitios Internos de Entrada al Ribosoma , Vacuna contra la Fiebre Amarilla/efectos adversos , Vacuna contra la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/genética , Antígenos Virales , Interferón Tipo I/genética , Mamíferos/genéticaRESUMEN
Landfill leachate has been documented as a significant source of trace organic pollutants, comprising an expansive family of per- and polyfluoroalkyl substances (PFAS). This study presents the findings on the distribution of 13 perfluoroalkyl carboxylates (PFCAs) and 4 perfluoroalkyl sulfonates (PFSAs) in leachates from 6 municipal solid waste (MSW) landfills in western China. The total concentrations of 17 PFAS in sampled leachates ranged from 1805 to 43,310 ng/L, and 15 compounds were detected in all samples. The short-chain compounds perfluorobutane sulfonate (PFBS, mean mass fraction 23.1%) and perfluorobutyric acid (PFBA, mean mass fraction 20.6%) were dominant. There were higher PFAS concentrations in leachates from operating landfills (mean: 12,194 ng/L) compared to closed landfills (mean: 2747 ng/L), but there was no significant difference between young (< 10 years) and old landfills (> 10 years). Moderate to weak correlations were observed between PFAS concentrations and leachate properties, e.g., TN, NH4+-N, TOC, and pH. This is the first report on the distribution of PFAS in landfill leachates from western China. The results have identified landfill leachate as an underestimated source of PFAS in the environment and have contributed to a more comprehensive evaluation on PFAS presence across China.
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Fluorocarburos , Eliminación de Residuos , Contaminantes Químicos del Agua , China , Monitoreo del Ambiente/métodos , Fluorocarburos/análisis , Residuos Sólidos/análisis , Instalaciones de Eliminación de Residuos , Contaminantes Químicos del Agua/análisisRESUMEN
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.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , Ratones , Unión Proteica , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.
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COVID-19/complicaciones , COVID-19/virología , Glucosa/metabolismo , Hiperglucemia/etiología , Hiperglucemia/metabolismo , Proteínas de la Membrana/metabolismo , SARS-CoV-2 , Animales , Biomarcadores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ayuno , Expresión Génica , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Interacciones Huésped-Patógeno , Humanos , Hiperglucemia/sangre , Hígado/metabolismo , Hígado/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Especificidad de Órganos/genéticaAsunto(s)
Aminoácidos/genética , Codón de Terminación/genética , Código Genético/genética , Recombinación Genética , Virus Zika/genética , Animales , Secuencia de Bases , Chlorocebus aethiops , ADN Recombinante/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas/genética , Células Vero , Proteínas Virales/genética , Replicación Viral/genética , Virus Zika/fisiología , Infección por el Virus Zika/virologíaRESUMEN
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.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19 , Epítopos/inmunología , SARS-CoV-2/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19/inmunología , COVID-19/prevención & control , Chlorocebus aethiops , Modelos Animales de Enfermedad , Humanos , Anticuerpos de Cadena Única/farmacología , Células VeroRESUMEN
There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this 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 (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a 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 is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.
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ARN Mensajero/genética , ARN Viral/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunogenicidad Vacunal , Inyecciones Intramusculares , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Nanopartículas/química , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células TH1/inmunología , Potencia de la Vacuna , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
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 an 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) by using CRISPR/Cas9 knockin technology. In comparison 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 seen 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.
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Betacoronavirus/fisiología , Infecciones por Coronavirus , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Pandemias , Neumonía Viral , Envejecimiento , Enzima Convertidora de Angiotensina 2 , Animales , Encéfalo/virología , COVID-19 , Sistemas CRISPR-Cas , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Citocinas/sangre , Técnicas de Sustitución del Gen , Pulmón/patología , Pulmón/virología , Enfermedades Pulmonares Intersticiales/patología , Nariz/virología , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , ARN Viral/análisis , SARS-CoV-2 , Estómago/virología , Tráquea/virología , Carga Viral , Replicación ViralRESUMEN
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 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-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.