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.

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.

Background: Pathogenic flaviviruses pose a serious threat to human health, and vaccines are an effective means of prevention and control. Although related vaccines have achieved significant progress, safety and efficacy limitations still exist, urgently requiring the development of novel vaccine platforms. The insect-specific flavivirus Chaoyang virus (CYV), with a structure similar to pathogenic flaviviruses and limited to insect cell replication, has potential as a safe vaccine vector. Methods: To systematically evaluate CYV’s potential as a universal flavivirus vaccine backbone and provide a vaccine candidate for type I Japanese encephalitis virus (JEV) prevention, this study constructed a chimeric JEV genotype I (GI) prME protein vaccine candidate CYV-JEV using CPER technology, systematically assessing its safety and immunoprotective effects. Results: Using the CPER method, CYV-JEV was successfully rescued, showing efficient replication in mosquito cells but defective replication in mammalian cells. As a vaccine backbone, CYV did not induce inflammatory responses or immune cell subset imbalances in IFNAR^−/− mice. CYV-JEV exhibited no pathogenicity in adult and suckling IFNAR^−/− mice. Immunisation of IFNAR^−/− mice with 10⁶ FFU twice provided complete protection against lethal challenge (100%) and effectively reduced paralysis rates (62.5%). Single-cell sequencing further revealed extensive T- and B-cell activation in the immune spleen. Conclusions: The results demonstrate that the CYV-based CYV-JEV candidate vaccine demonstrates both safety and efficacy, representing a promising alternative to attenuated JEV vaccines, with CYV showing potential as a safe and effective universal flavivirus vaccine backbone.