Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2-3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.

Some rodent-borne hantaviruses are known to cause hemorrhagic fever with renal syndrome in Europe and Asia, and hantavirus cardiopulmonary syndrome in the Americas. Despite the significant public health threat caused by hantaviruses, there are no antiviral therapeutics approved to treat hantavirus infections. One of the major limitations to study these viruses is the requirement for biosafety level 3 (BSL-3) containment. To address this concern, we previously generated a Seoul virus (SEOV) minigenome system which could be used to screen antivirals at BSL-2 level. Here, we report the development of a similar minigenome system based on the L segment of the prototype hantavirus, Hantaan virus (HTNV). In addition, we examined the activity of minigenomes based on M and S segments of SEOV and HTNV. Furthermore, we used the new HTNV minigenome system to confirm the activity of a selected group of antiviral compounds targeting the viral polymerase. All tested compounds (2'-deoxy-2'-Fluorocytidine, baloxavir, remdesivir and ribavirin) show potent anti-HTNV activity. The minigenome systems could be useful tools to study replication mechanisms and to screen antiviral compounds against hantaviruses at lower containment laboratories.

The quantification of biological assays, such as plaque and microbial assays is essential in virology and microbiology research. However, low-contrast images of stain-free samples are difficult to segment accurately and manual labeling is time-consuming. To address these problems, we present a weakly supervised framework for automated biological assay assessment. First, we collected and constructed weakly supervised datasets for viral plaque and microbial colony segmentation using point and bounding box annotations respectively. Then, we proposed an adaptive region-growing algorithm that generates mask annotations, reducing annotation burden. We adapted and fine-tuned automatic Segment Anything Model (SAM) to for biological specimen segmentation, demonstrating improved accuracy across diverse assay types. Moreover, we also validated our method on live cell segmentation. Finally, we applied our model in antiviral compound assessment and achieved comparable results to manual assessment. In summary, our framework provides an efficient and automated solution for biological assay quantification, reducing annotation burden while maintaining accuracy.