The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital syndromes including microcephaly and fetal demise in pregnant women. No commercial vaccines against ZIKV are currently available. We previously generated a chimeric ZIKV (ChinZIKV) based on the Chaoyang virus (CYV) by replacing the prME protein of CYV with that of a contemporary ZIKV strain GZ01. Herein, we evaluated this vaccine candidate in a mouse model and showed that ChinZIKV was totally safe in both adult and suckling immunodeficient mice. No viral RNA was detected in the serum of mice inoculated with ChinZIKV. All of the mice inoculated with ChinZIKV survived, while mice inoculated with ZIKV succumbed to infection in 8 days. A single dose of ChinZIKV partially protected mice against lethal ZIKV challenge. In contrast, all the control PBS-immunized mice succumbed to infection after ZIKV challenge. Our results warrant further development of ChinZIKV as a vaccine candidate in clinical trials.

Pathogenic flaviviruses are predominantly the pathogens of emerging and re-emerging infectious diseases, which have caused multiple public health emergencies globally and pose a serious threat to human health and social development. Although significant achievements have been made in vaccine research, issues such as limited protective effects and virulence reversion persist, making the development of novel vaccines against pathogenic flaviviruses a current research hotspot and challenge. ISFVs have recently attracted attention due to their high homology with pathogenic flaviviruses and unique inability to replicate in mammalian hosts. Multiple vaccine candidate strains constructed using ISFVs as scaffolds have demonstrated excellent safety and efficacy. This review summarizes the biological characteristics, host restriction factors, current applications in vaccine development, and challenges faced by ISFVs, providing a reference for future research on pathogenic flavivirus vaccines.

The vaccinia virus (VV) is extensively utilized as a vaccine vector in the treatment of various infectious diseases, cardiovascular diseases, immunodeficiencies, and cancers. The vaccinia virus Tiantan strain (VVTT) has been instrumental as an irreplaceable vaccine strain in the eradication of smallpox in China; however, it still presents significant adverse toxic effects. After the WHO recommended that routine smallpox vaccination be discontinued, the Chinese government stopped the national smallpox vaccination program in 1981. The outbreak of monkeypox in 2022 has focused people’s attention on the Orthopoxvirus. However, there are limited reports on the safety and toxic side effects of VVTT. In this study, we employed a combination of transcriptomic analysis and machine learning-based feature selection to identify key genes implicated in the VVTT infection process. We utilized four machine learning algorithms, including random forest (RF), minimum redundancy maximum relevance (MRMR), eXtreme Gradient Boosting (XGB), and least absolute shrinkage and selection operator cross-validation (LASSOCV), for feature selection. Among these, XGB was found to be the most effective and was used for further screening, resulting in an optimal model with an ROC curve of 0.98. Our analysis revealed the involvement of pathways such as spinocerebellar ataxia and the p53 signaling pathway. Additionally, we identified three critical targets during VVTT infection—ARC, JUNB, and EGR2—and further validated these targets using qPCR. Our research elucidates the mechanism by which VVTT infects cells, enhancing our understanding of the smallpox vaccine. This knowledge not only facilitates the development of new and more effective vaccines but also contributes to a deeper comprehension of viral pathogenesis. By advancing our understanding of the molecular mechanisms underlying VVTT infection, this study lays the foundation for the further development of VVTT. Such insights are crucial for strengthening global health security and ensuring a resilient response to future pandemics.

Background: Uricase, or urate oxidase (Uox) is a key enzyme in uric acid (UA) metabolism and has been applied in clinical treatment of human hyperuricemia (HUA). However, the current clinically applied uricases, despite their potent urate-lowering capacity, tend to form anti-drug antibodies because of their immunogenicity, leading to increased risk of anaphylaxis, faster drug clearance and reduced or even complete loss of therapeutic effect, limiting their clinical application. In this study, we constructed engineered macrophages that stably expressed uricase, which might serve as a promising alternative to the direct injection of uricases. Materials and Methods: Engineered macrophages RAW264.7 cells were injected intravenously to treat hyperuricemic KM mice. Serum uric acid and bio-indicators for renal and hepatic functions were detected by an automatic biochemical analyzer; inflammatory cytokines were determined by ELISA; the livers and kidneys of the mice were sectioned for histological examination. Results: The uricase-expressing macrophages reduced UA levels from 300 ± 1.5 μmol/L to 101 ± 8.3 μmol/L in vitro. And in an HUA mouse model established by gavage with yeast extract, intravenous injection of the engineered macrophages could reduce the serum uric acid (sUA) of mice to normal level on the 14th day of modeling, with a decrease of 48.6%, and the urate-lowering effect was comparable to that of the first-line clinical drug allopurinol. In terms of safety, engineered macrophages did not cause liver or kidney dysfunction in mice, nor did they induce systemic immune response. Conclusions: Using macrophages as a chassis to deliver uricase might be a new, safe and effective strategy for the treatment and control of hyperuricemia.

Reverse genetics is a useful tool for studying viruses and developing vaccines for coronaviruses. However, constructing and manipulating the coronavirus genome in Escherichia coli can be time-consuming and challenging due to its large size and instability. Homologous recombination, a genetic manipulation mechanism found in organisms, is essential for DNA repair, gene recombination, and genetic engineering. In yeast, particularly Saccharomyces cerevisiae, homologous recombination technology is commonly used for constructing gene expression plasmids and genome editing. In this study, we successfully split and spliced a 30 kb viral genome fragment using yeast homologous recombination. By optimizing the program parameters, such as homologous arm lengths and fragment-to-vector ratios, we achieved a splicing efficiency of up to 97.9%. The optimal parameters selected were a 60 bp homologous sequence size and a vector fragment ratio of 1:2:2:2:2:2 for yeast homologous recombination of large DNA fragments.