Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for ...SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.
Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA ...(cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.
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.
•Dengue virus NS5 methyltransferase and polymerase are well characterized biochemically, structurally and functionally.•Small molecular-weight compounds that inhibit its enzymatic activities and ...virus replication in cells have been reported.•Re-positioning of marketed drugs is also being explored for anti-dengue therapy.•Inhibitors against the polymerase are more tractable than those against the methyltransferase.•Nucleoside inhibitors against the polymerase have made the most progress, with one entering the clinic.
The non-structural protein 5 (NS5) of flaviviruses is the most conserved amongst the viral proteins. It is about 900kDa and bears enzymatic activities that play vital roles in virus replication. Its N-terminal domain encodes dual N7 and 2′-O methyltransferase activities (MTase), and possibly guanylyltransferase (GTase) involved in RNA cap formation. The C-terminal region comprises a RNA-dependent RNA polymerase (RdRp) required for viral RNA synthesis. Both MTase and RdRp activities of dengue virus NS5 are well characterized, structurally and functionally. Numerous crystal structures of the flavivirus MTase and RdRp domains have been solved. Inhibitors of both functions have been identified through screening activities using biochemical and cell-based assays, as well as via rational design approaches. This review summaries the current knowledge as well as prospective views on these aspects. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research.
The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection ...and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.
Recently, hydrogel-based flexible sensors have attracted tremendous attention for use in wearable soft electronics. However, under sub-zero temperatures, common hydrogel-based flexible devices are ...always out of work due to their poor stability in freezing environments. To endow hydrogel-based sensors with long-term stability and anti-freezing ability as well as multi-functional abilities, we developed a polydopamine-reduced graphene oxide (PDA-rGO)/sodium alginate (SA)/polyacrylamide (PAM) composite organohydrogel with dual crosslinking networks. The excellent conductivity of the organohydrogel is due to the well-dispersed rGO endowed by mussel-inspired chemistry and ions such as Ca
2+
, which give the organohydrogel strain, pressure, and temperature sensing capabilities with a high gauge factor (2.09) within a broad strain range (0-250%), short response time (200 ms), and a wide temperature detection range (−20 °C to 60 °C), respectively. Moreover, the assembled sensors can also detect multiple human motions such as finger bending, facial micro-expression, and hand gesture recognition. Especially, owing to the synergistic effects of ion transportation, water-glycerol binary solvent, and the reduced graphene oxide in the composite hydrogel, the organohydrogel achieved an unprecedented thermal sensitivity of 97.60% °C
−1
at sub-zero temperatures, which is the most sensitive stretchable thermistor so far reported. Therefore, this as-prepared functional organohydrogel paves the way for potential applications in human-machine interactions and personalized multi-signal monitoring in a broad temperature range.
The excellent properties and the multiple sensing applications of the PDA-rGO/SA/PAM organohydrogel.
The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of ...SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.
The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.
A total of 20 serum samples from 15 persons who received the BNT162b2 vaccine showed strong neutralization activity against recombinant viruses engineered to express the spike protein from the ...Wuhan-Hu-1 strain and B.1.429, B.1.526, and B.1.1.7 variants. These in vitro neutralization assays are not clearly predictive of activity in vivo.
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