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PubMed2026

Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus for which no specific antiviral therapy is currently available. During the large outbreak in Foshan, Guangdong Province, China, in July 2025, CHIKV rapidly spread to neighboring regions and caused more than 16,000 confirmed cases. In this study, the predominant outbreak strain of CHIKV was selected as the reference sequence to establish a panel of complementary biosafe tools for antiviral compound screening and mechanistic investigation. A virus replicon particle (VRP) system for CHIKV was first constructed and applied to compound library screening, resulting in the identification of three candidate antiviral compounds: MDL-12330A, bazedoxifene acetate, and anidulafungin. To further validate their antiviral activities and investigate their potential mechanisms, CHIKV functional evaluation systems were subsequently established, including vesicular stomatitis virus (VSV)- and murine leukemia virus (MLV)-based pseudovirus systems for viral entry, a replicon RNA system for post-entry replication-associated processes, a replication-defective nsP4 mutant replicon RNA system for primary translation, and a virus-like particle (VLP) system for viral particle assembly and budding assessment. Using these complementary systems, we systematically evaluated the antiviral profiles of the three candidate compounds across multiple stages of the CHIKV life cycle. This analysis revealed distinct stage-specific inhibitory patterns and provided insights into their potential antiviral mechanisms, which warrant validation using authentic CHIKV infection to assess their translational potential.

Chikungunya virus↗

PubMed2025

Reverse Genetics System for Crimean-Congo Hemorrhagic Fever Virus.

Reverse genetic systems are powerful tools in molecular virology that allow the generation of infectious recombinant virus and the manipulation of viral genomes. Reverse genetic systems enable the incorporation of reporter genes, facilitating many virological assays, including high-throughput screening. Additionally, reverse genetic systems can be used to introduce targeted mutations into the viral genome, allowing investigations of viral genetic elements and protein functions in virus pathogenesis and biology. Here we describe in detail the materials and methods required for the Crimean-Congo hemorrhagic fever virus (CCHFV) reverse genetic system. This system can be used to generate complete infectious recombinant virus, and virus-like replicon particles (VRPs) lacking the M segment but complemented with an exogenous source of glycoprotein precursor (GPC); resulting in single-round replicon particles that can be used to study components of the viral replicative cycle at a lower biosafety level.

Hemorrhagic Fever Virus, Crimean-Congo↗

PubMed2024

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Animals↗

PubMed1978

Construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.

A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.

Bacterial Proteins↗

PubMed1978

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids".

A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

Coliphages↗