An accumulated mutation gained in mosquito cells enhances Zika virus virulence and fitness in mice.

Zika virus (ZIKV) remains a significant public health threat worldwide. A number of adaptive mutations have accumulated within the genome of ZIKV during global transmission, some of which have been linked to specific phenotypes. ZIKV maintains an alternating cycle of replication between mosquitoes and vertebrate hosts, but the role of mosquito-specific adaptive mutations in ZIKV has not been well investigated. In this study, we demonstrated that serial passaging of ZIKV in mosquito Aag2 cells led to the emergence of critical amino acid substitutions, including A94V in the prM protein and V153D and H401Y in the E protein. Further characterization via reverse genetics revealed that the H401Y substitution in the E protein did not augment viral replication in mosquitoes but significantly enhanced neurovirulence and lethality compared with those of the wild-type (WT) virus in mice. More importantly, the H401Y mutant maintained its virulence phenotype in mice after propagation in mosquitoes in mosquito-mouse cycle model. In particular, recombinant ZIKV harboring the H401Y substitution showed enhanced competitive fitness over WT ZIKV in various mammalian cells and mouse brains, but not in mosquito cells. Notably, the H401Y substitution in the ZIKV E protein has been detected in recent isolates derived from both mosquitoes and humans in Asia and the Americas. In summary, our findings not only identify a novel virulence determinant of ZIKV but also highlight the complexity of the relationship between the evolution of vector-borne viruses and their clinical outcome in nature. IMPORTANCE: Zika virus (ZIKV) is an important arbovirus with a global impact. Experimental evolution by serial passaging of ZIKV in susceptible cells has led to the identification of a panel of critical amino acid substitutions with specific functions. Herein, we identified a mosquito cell-derived substitution, H401Y, in the ZIKV E protein via experimental evolution. The H401Y substitution significantly enhanced viral virulence and fitness in mammal cells and mice. Notably, the H401Y substitution has been detected in recent mosquito and human isolates from regions spanning Asia to the Americas. Our work elucidates unrecognized virulence determinant in the ZIKV genome that warrants urgent attention. Moreover, the findings underscore the critical need for extensive molecular surveillance and rigorous clinical observation to establish the potential impact in natural circulation. These endeavors are crucial for unraveling the potential of mutation to act as a catalyst for future epidemics, thereby preempting the public health challenges it may pose.

Establishment of enterically transmitted hepatitis virus animal models using lipid nanoparticle-based full-length viral genome RNA delivery system.

BACKGROUND: Enterically transmitted hepatitis viruses, such as hepatitis A virus (HAV) and hepatitis E virus (HEV), remain notable threats to public health. However, stable and reliable animal models of HAV and HEV infection are lacking. OBJECTIVE: This study aimed to establish HAV and HEV infections in multiple small animals by intravenously injecting lipid nanoparticle (LNP)-encapsulated full-length viral RNAs (LNP-vRNA). DESIGN: In vitro transcribed and capped full-length HAV RNA was encapsulated into LNP and was intravenously inoculated to Ifnar-/- mice, and HEV RNA to rabbits and gerbils. Virological parameters were determined by RT-qPCR, ELISA and immunohistochemistry. Liver histopathological changes were analysed by H&E staining. Antiviral drug and vaccine efficacy were further evaluated by using the LNP-vRNA-based animal model. RESULTS: On intravenous injection of LNP-vRNA, stable viral shedding was detected in the faeces and infectious HAV or HEV was recovered from the livers of the inoculated animals. Liver damage was observed in LNP-vRNA (HAV)-injected mice and LNP-vRNA (HEV)-injected rabbits. Mongolian gerbils were also susceptible to LNP-vRNA (HEV) injections. Finally, the antiviral countermeasures and in vivo function of HEV genome deletions were validated in the LNP-vRNA-based animal model. CONCLUSION: This stable and standardised LNP-vRNA-based animal model provides a powerful platform to investigate the pathogenesis and evaluate countermeasures for enterically transmitted hepatitis viruses and can be further expanded to other viruses that are not easily cultured in vitro or in vivo.

Harnessing ZIKV NS2A RNA for alleviating acute hepatitis and cytokine release storm by targeting translation machinery.

BACKGROUND AND AIMS: Hyperactivated inflammatory responses induced by cytokine release syndrome are the primary causes of tissue damage and even death. The translation process is precisely regulated to control the production of proinflammatory cytokines. However, it is largely unknown whether targeting translation can effectively limit the hyperactivated inflammatory responses during acute hepatitis and graft-versus-host disease. APPROACH AND RESULTS: By using in vitro translation and cellular overexpression systems, we have found that the nonstructural protein gene NS2A of Zika virus functions as RNA molecules to suppress the translation of both ectopic genes and endogenous proinflammatory cytokines. Mechanistically, results from RNA pulldown and co-immunoprecipitation assays have demonstrated that NS2A RNA interacts with the translation initiation factor eIF2α to disrupt the dynamic balance of the eIF2/eIF2B complex and translation initiation, which is the rate-limiting step of translation. In the acetaminophen-induced, lipopolysaccharide/D-galactosamine-induced, viral infection-induced acute hepatitis, and graft-versus-host disease mouse models, mice with myeloid cell-specific knock-in of NS2A show decreased levels of serum proinflammatory cytokines and reduced tissue damage. CONCLUSIONS: Zika virus NS2A dampens the production of proinflammatory cytokines and alleviates inflammatory injuries by interfering translation process as RNA molecules, which suggests that NS2A RNA is potentially used to treat numerous acute inflammatory diseases characterized by cytokine release syndrome.

Rational design of a DNA-launched live attenuated vaccine against human enterovirus 71.

Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/ßR-/- mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 â€‹µg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.

SARS-CoV-2 Evolution: Immune Dynamics, Omicron Specificity, and Predictive Modeling in Vaccinated Populations.

Host immunity is central to the virus's spread dynamics, which is significantly influenced by vaccination and prior infection experiences. In this work, we analyzed the co-evolution of SARS-CoV-2 mutation, angiotensin-converting enzyme 2 (ACE2) receptor binding, and neutralizing antibody (NAb) responses across various variants in 822 human and mice vaccinated with different non-Omicron and Omicron vaccines is analyzed. The link between vaccine efficacy and vaccine type, dosing, and post-vaccination duration is revealed. The classification of immune protection against non-Omicron and Omicron variants is co-evolved with genetic mutations and vaccination. Additionally, a model, the Prevalence Score (P-Score) is introduced, which surpasses previous algorithm-based models in predicting the potential prevalence of new variants in vaccinated populations. The hybrid vaccination combining the wild-type (WT) inactivated vaccine with the Omicron BA.4/5 mRNA vaccine may provide broad protection against both non-Omicron variants and Omicron variants, albeit with EG.5.1 still posing a risk. In conclusion, these findings enhance understanding of population immunity variations and provide valuable insights for future vaccine development and public health strategies.

Identification of mutations in viral proteins involved in cell adaptation using a reverse genetic system of the live attenuated hepatitis A virus vaccine H2 strain.

The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures. Currently, the mechanism underlying its attenuation phenotype remain largely unknown. In this study, we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques. The recovered H2 strain (H2ic) from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production, similar to the parental H2 strain. Additionally, H2ic did not cause disease in Ifnar1-/- C57 mice, consistent with the H2 strain. To explore the cell-adaptive mutations of the H2 strain, chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone. The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic. Other chimeric viruses containing the 2B, 2C, or 3A proteins from H2w failed to be recovered. Furthermore, there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses. These results demonstrate that adaptive mutations in the 2B, 2C, and 3A proteins are essential for efficient replication of the H2 strain in cell cultures. Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice. Together, this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.

Immunogenicity and Protective Efficacy of Aerosolized Live-Attenuated Yellow Fever 17D Vaccine in Mice.

Yellow fever (YF), caused by the yellow fever virus (YFV), continually spreads and causes epidemics worldwide, posing a great threat to human health. The live-attenuated YF 17D vaccine (YF-17D) has been licensed for preventing YFV infection and administrated via the intramuscular (i.m.) route. In this study, we sought to determine the immunogenicity and protective efficacy of aerosolized YF-17D via the intratracheal (i.t.) route in mice. YF-17D stocks in liquids were successfully aerosolized into particles of 6 µm. Further in vitro phenotype results showed the aerosolization process did not abolish the infectivity of YF-17D. Meanwhile, a single i.t. immunization with aerosolized YF-17D induced robust humoral and cellular immune responses in A129 mice, which is comparable to that received i.p. immunization. Notably, the aerosolized YF-17D also triggered specific secretory IgA (SIgA) production in bronchoalveolar lavage. Additionally, all immunized animals survived a lethal dose of YFV challenge in mice. In conclusion, our results support further development of aerosolized YF-17D in the future.

Efficacy, immunogenicity, and safety of a monovalent mRNA vaccine, ABO1020, in adults: A randomized, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: ABO1020 is a monovalent COVID-19 mRNA vaccine. Results from a phase 1 trial showed ABO1020 was safe and well tolerated, and phase 3 trials to evaluate the efficacy, immunogenicity, and safety of ABO1020 in healthy adults are urgently needed. METHODS: We conducted a multinational, randomized, placebo-controlled, double-blind, phase 3 trial among healthy adults (ClinicalTrials.gov: NCT05636319). Participants were randomly assigned (1:1) to receive either 2 doses of ABO1020 (15 µg per dose) or placebo, administered 28 days apart. The primary endpoint was the vaccine efficacy in preventing symptomatic COVID-19 cases that occurred at least 14 days post-full vaccination. The second endpoint included the neutralizing antibody titers against Omicron BA.5 and XBB and safety assessments. FINDINGS: A total of 14,138 participants were randomly assigned to receive either vaccine or placebo (7,069 participants in each group). A total of 366 symptomatic COVID-19 cases were confirmed 14 days after the second dose among 93 participants in the ABO1020 group and 273 participants in the placebo group, yielding a vaccine efficacy of 66.18% (95% confidence interval: 57.21-73.27, p < 0.0001). A single dose or two doses of ABO1020 elicited potent neutralizing antibodies against both BA.5 and XBB.1.5. The safety profile of ABO1020 was characterized by transient, mild-to-moderate fever, pain at the injection site, and headache. CONCLUSION: ABO1020 was well tolerated and conferred 66.18% protection against symptomatic COVID-19 in adults. FUNDING: National Key Research and Development Project of China, Innovation Fund for Medical Sciences from the CAMS, National Natural Science Foundation of China.

Atavistic strategy for the treatment of hyperuricemia via ionizable liposomal mRNA.

Hyperuricemia is associated with an increased risk of gout, hypertension, diabetes, and cardiovascular diseases. Most mammals maintain normal serum uric acid (SUA) via urate oxidase (Uox), an enzyme that metabolizes poorly-soluble UA to highly-soluble allantoin. In contrast, Uox became a pseudogene in humans and apes over the long course of evolution. Here we demonstrate an atavistic strategy for treating hyperuricemia based on endogenous expression of Uox in hepatocytes mediated by mRNA (mUox) loaded with an ionizable lipid nanoparticle termed iLAND. mUox@iLAND allows effective transfection and protein expression in vitro. A single dose of mUox@iLAND lowers SUA levels for several weeks in two female murine models, including a novel long-lasting model, which is also confirmed by metabolomics analysis. Together with the excellent safety profiles observed in vivo, the proposed mRNA agent demonstrates substantial potential for hyperuricemia therapy and the prevention of associated conditions.

Severe Organ Impairment Was Common in Elderly Individuals with Dengue in Guangdong, China.

Guangdong, China, has experienced several dengue epidemics involving thousands of confirmed cases in recent decades, and elderly individuals suffered severe dengue (SD) most seriously. However, the clinical characteristics and risk factors for SD among elderly patients in Guangdong have not been investigated. Patients older than 65 years were recruited and divided into a dengue fever (DF) group and an SD group according to the 2009 Dengue Guidelines of the WHO. We analyzed the clinical manifestations of the elderly patients with dengue and then assessed the risk factors for SD. Of a total of 1,027 patients, 868 patients were diagnosed as having DF and 159 as having SD. Of the 159 elderly patients with SD, 129 (81%) had comorbidities, with hypertension being the most common. Severe organ impairment (SOI) (115, 54%) was the most common presentation in SD patients, followed by severe plasma leakage (52, 24.4%) and severe hemorrhage (46, 21.6%). The most common symptom of SOI was kidney injury, followed by heart injury and central nervous system injury. Furthermore, multivariate regression revealed that the presence of chronic obstructive pulmonary disease (COPD), a lower red blood cell (RBC) count (≤3.5 × 1012/L; odds ratio [OR], 0.35; 95% CI, 0.17-0.55; P <0.001), lower serum albumin (ALB) (≤35 U/L; OR, 0.18; 95% CI, 0.09-0.32; P <0.001), and hyperpyrexia (body temperature ≥39°C; OR, 1.8; 95% CI, 1.2-2.6, P <0.001) were risk factors for SD. Severe organ impairment was the predominant manifestation in elderly individuals with SD characterized by kidney injury. The potential risk factors of SD such as presence of COPD and hyperpyrexia and lower RBC and ALB levels might help clinicians identify patients with SD early.

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments.

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

Peripheral nerve injury associated with JEV infection in high endemic regions, 2016-2020: a multicenter retrospective study in China.

Previously, we reported a cohort of Japanese encephalitis (JE) patients with Guillain-Barré syndrome. However, the evidence linking Japanese encephalitis virus (JEV) infection and peripheral nerve injury (PNI) remains limited, especially the epidemiology, clinical presentation, diagnosis, treatment, and outcome significantly differ from traditional JE. We performed a retrospective and multicenter study of 1626 patients with JE recorded in the surveillance system of the Chinese Center for Disease Control and Prevention, spanning the years 2016-2020. Cases were classified into type 1 and type 2 JE based on whether the JE was combined with PNI or not. A comparative analysis was conducted on demographic characteristics, clinical manifestations, imaging findings, electromyography data, laboratory results, and treatment outcomes. Among 1626 laboratory confirmed JE patients, 230 (14%) were type 2 mainly located along the Yellow River in northwest China. In addition to fever, headache, and disturbance of consciousness, type 2 patients experienced acute flaccid paralysis of the limbs, as well as severe respiratory muscle paralysis. These patients presented a greater mean length of stay in hospital (children, 22 years [range, 1-34]; adults, 25 years [range, 0-183]) and intensive care unit (children, 16 years [range, 1-30]; adults, 17 years [range, 0-102]). The mortality rate was higher in type 2 patients (36/230 [16%]) compared to type 1 (67/1396 [5%]). The clinical classification of the diagnosis of JE may play a crucial role in developing a rational treatment strategy, thereby mitigating the severity of the disease and potentially reducing disability and mortality rates among patients.

The infection kinetics and transmission potential of two Guaico Culex viruses in Culex quinquefasciatus mosquitoes.

Guaico Culex virus (GCXV) is a newly identified segmented Jingmenvirus from Culex spp. mosquitoes in Central and South America. The genome of GCXV is composed of four or five single-stranded positive RNA segments. However, the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown. In this study, we used reverse genetics to rescue two GCXVs (4S and 5S) that contained four and five RNA segments, respectively, in C6/36 â€‹cells. Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics, protein expression and viral titers. Importantly, GCXV RNAs were detected in the bodies, salivary glands, midguts and ovaries of Culex quinquefasciatus at 4-10 days after oral infection. In addition, two GCXVs can colonize Cx. quinquefasciatus eggs, resulting in positive rates of 15%-35% for the second gonotrophic cycle. In conclusion, our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx. quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.

Rapid development of double-hit mRNA antibody cocktail against orthopoxviruses.

The Orthopoxvirus genus, especially variola virus (VARV), monkeypox virus (MPXV), remains a significant public health threat worldwide. The development of therapeutic antibodies against orthopoxviruses is largely hampered by the high cost of antibody engineering and manufacturing processes. mRNA-encoded antibodies have emerged as a powerful and universal platform for rapid antibody production. Herein, by using the established lipid nanoparticle (LNP)-encapsulated mRNA platform, we constructed four mRNA combinations that encode monoclonal antibodies with broad neutralization activities against orthopoxviruses. In vivo characterization demonstrated that a single intravenous injection of each LNP-encapsulated mRNA antibody in mice resulted in the rapid production of neutralizing antibodies. More importantly, mRNA antibody treatments showed significant protection from weight loss and mortality in the vaccinia virus (VACV) lethal challenge mouse model, and a unique mRNA antibody cocktail, Mix2a, exhibited superior in vivo protection by targeting both intracellular mature virus (IMV)-form and extracellular enveloped virus (EEV)-form viruses. In summary, our results demonstrate the proof-of-concept production of orthopoxvirus antibodies via the LNP-mRNA platform, highlighting the great potential of tailored mRNA antibody combinations as a universal strategy to combat orthopoxvirus as well as other emerging viruses.

Development of a live-attenuated chimeric vaccine against the emerging Usutu virus.

Usutu virus (USUV) is an emerging arthropod-borne flavivirus that has expanded into multiple European countries during the past several decades. USUV infection in human has been linked to severe neurological complications, and no vaccine is now available against USUV. In this work, we develop a live-attenuated chimeric USUV vaccine (termed ChinUSUV) based on the full-length infectious cDNA clone of the licensed Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. In vitro studies demonstrate that ChinUSUV replicates efficiently and maintains its genetic stability. Remarkably, ChinUSUV exhibits a significant attenuation phenotype in multiple mouse models even compared with the licensed JEV vaccine. A single immunization with ChinUSUV elicits potent IgG and neutralizing antibody responses as well as T cell response. Passive transfer of sera from ChinUSUV-immunized mice confers significant protection against lethal homologous challenge in suckling mice. Taken together, our results suggest that ChinUSUV represents a potential USUV vaccine candidate that merits further development.

The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components.

During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

The aldehyde dehydrogenase ALDH1B1 exerts antiviral effects through the aggregation of the adaptor MAVS.

Type I interferons (IFNs) are produced by almost all cell types and play a vital role in host defense against viral infection. Infection with an RNA virus activates receptors such as RIG-I, resulting in the recruitment of the adaptor protein MAVS to the RIG-I-like receptor (RLR) signalosome and the formation of prion-like functional aggregates of MAVS, which leads to IFN-ß production. Here, we identified the aldehyde dehydrogenase 1B1 (ALDH1B1) as a previously uncharacterized IFN-stimulated gene (ISG) product with critical roles in the antiviral response. Knockout of ALDH1B1 increased, whereas overexpression of ALDH1B1 restricted, the replication of RNA viruses, such as vesicular stomatitis virus (VSV), Zika virus (ZIKV), dengue virus (DENV), and influenza A virus (IAV). We found that ALDH1B1 localized to mitochondria, where it interacted with the transmembrane domain of MAVS to promote MAVS aggregation. ALDH1B1 was recruited to MAVS aggregates. In addition, ALDH1B1 also enhanced the interaction between activated RIG-I and MAVS, thus increasing IFN-ß production and the antiviral response. Furthermore, Aldh1b1-/- mice developed more severe symptoms than did wild-type mice upon IAV infection. Together, these data identify an aldehyde dehydrogenase in mitochondria that functionally regulates MAVS-mediated signaling and the antiviral response.

A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing Group II Intron.

Zika virus (ZIKV) is a mosquito-borne flavivirus of the Flaviviridae family first isolated from a sentinel monkey in the Zika Forest, Uganda, in 1947. Since 2007, the virus has had a vast geographic expansion that extended to the Americas in 2015, leading to a series of large outbreaks. Although mainly transmitted by the bite of Aedes mosquitoes, human infection of ZIKV can also happen through unconventional routes such as sexual intercourse and, more importantly, vertical transmission. The genome of ZIKV is a single-stranded, positive-sense RNA molecule about 11 kb in length. The genome contains a single opening reading frame (ORF) flanked by highly structured 5' and 3' untranslated regions. To understand the mechanisms about ZIKV replication, transmission, and pathogenesis, reverse genetic tools are of great importance. In this chapter, a novel system is described for the generation and manipulation of a ZIKV infectious clone stabilized by a self-splicing group II intron, a mobile element with ribozyme activity. The intron can be spliced in vitro, and thus full-length vRNA can be prepared allowing virus genome manipulation required for further studies.

Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern.

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.