Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Platycodin D2 enhances P21/CyclinA2-mediated senescence of HCC cells by regulating NIX-induced mitophagy.

BACKGROUND: Hepatocellular carcinoma (HCC) cells usually show strong resistance to chemotherapy, which not only reduces the efficacy of chemotherapy but also increases the side effects. Regulation of autophagy plays an important role in tumor treatment. Cell senescence is also an important anti-cancer mechanism, which has become an important target for tumor treatment. Therefore, it is of great clinical significance to find anti-HCC drugs that act through this new mechanism. Platycodin D2 (PD2) is a new saponin compound extracted from the traditional Chinese medicine Platycodon grandiflorum. PURPOSE: Our study aimed to explore the effects of PD2 on HCC and identify the underlying mechanisms. METHODS: First, the CCK8 assay was used to detect the inhibitory effect of PD2 on HCC cells. Then, different pathways of programmed cell death and cell cycle regulators were measured. In addition, we assessed the effects of PD2 on the autophagy and senescence of HCC cells by flow cytometry, immunofluorescence staining, and Western blotting. Finally, we studied the in vivo effect of PD2 on HCC cells by using a mouse tumor-bearing model. RESULTS: Studies have shown that PD2 has a good anti-tumor effect, but the specific molecular mechanism has not been clarified. In this study, we found that PD2 has no obvious toxic effect on normal hepatocytes, but it can significantly inhibit the proliferation of HCC cells, induce mitochondrial dysfunction, enhance autophagy and cell senescence, upregulate NIX and P21, and downregulate CyclinA2. Gene silencing and overexpression indicated that PD2 induced mitophagy in HCC cells through NIX, thereby activating the P21/CyclinA2 pathway and promoting cell senescence. CONCLUSIONS: These results indicate that PD2 induces HCC cell death through autophagy and aging. Our findings provide a new strategy for treating HCC.

Codon Usage Bias Analysis of Bluetongue Virus Causing Livestock Infection.

Bluetongue virus (BTV) is a double-stranded RNA virus with multiple segments and belongs to the genus Orbivirus within the family Reoviridae. BTV is spread to livestock through its dominant vector, biting midges of genus Culicoides. Although great progress has been made in genomic analyses, it is not fully understood how BTVs adapt to their hosts and evade the host's immune systems. In this study, we retrieved BTV genome sequences from the National Center for Biotechnology Information (NCBI) database and performed a comprehensive research to explore the codon usage patterns in 50 BTV strains. We used bioinformatic approaches to calculate the relative synonymous codon usage (RSCU), codon adaptation index (CAI), effective number of codons (ENC), and other indices. The results indicated that most of the overpreferred codons had A-endings, which revealed that mutational pressure was the major force shaping codon usage patterns in BTV. However, the influence of natural selection and geographical factors cannot be ignored on viral codon usage bias. Based on the RSCU values, we performed a comparative analysis between BTVs and their hosts, suggesting that BTVs were inclined to evolve their codon usage patterns that were comparable to those of their hosts. Such findings will be conducive to understanding the elements that contribute to viral evolution and adaptation to hosts.

Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency.

The evolution of organisms has provided a variety of mechanisms to maintain the integrity of its genome, but as damage occurs, DNA damage repair pathways are necessary to resolve errors. Among them, the DNA double-strand break repair pathway is highly conserved in eukaryotes, including mammals. Nonhomologous DNA end joining and homologous directed repair are two major DNA repair pathways that are synergistic or antagonistic. Clustered regularly interspaced short palindromic repeats genome editing techniques based on the nonhomologous DNA end joining repair pathway have been used to generate highly efficient insertions or deletions of variable-sized genes but are error-prone and inaccurate. By combining the homology-directed repair pathway with clustered regularly interspaced short palindromic repeats cleavage, more precise genome editing via insertion or deletion of the desired fragment can be performed. However, homologous directed repair is not efficient and needs further improvement. Here, we describe several ways to improve the efficiency of homologous directed repair by regulating the cell cycle, expressing key proteins involved in homologous recombination and selecting appropriate donor DNA.

The Commensal Microbiota and Viral Infection: A Comprehensive Review.

The human body is inhabited by a diverse microbial community that is collectively coined as commensal microbiota. Recent research has greatly advanced our understanding of how the commensal microbiota affects host health. Among the various kinds of pathogenic infections of the host, viral infections constitute one of the most serious public health problems worldwide. During the infection process, viruses may have substantial and intimate interactions with the commensal microbiota. A plethora of evidence suggests that the commensal microbiota regulates and is in turn regulated by invading viruses through diverse mechanisms, thereby having stimulatory or suppressive roles in viral infections. Furthermore, the integrity of the commensal microbiota can be disturbed by invading viruses, causing dysbiosis in the host and further influencing virus infectivity. In the present article, we discuss current insights into the regulation of viral infection by the commensal microbiota. We also draw attention to the disruption of microbiota homeostasis by several viruses.

Pathogenicity of blood orf virus isolates in the development of dairy goat contagious pustular dermatitis.

Contagious pustular dermatitis is an exanthematous zoonotic disease caused by the orf virus. Pandemic outbreaks of this disease cause great economic losses, while the pathogenesis of this disease still remains obscure. In this study, blood samples were collected from 628 asymptomatic goats across China for PCR-based virus detection. We detected the orf virus in the blood of asymptomatic goats. Moreover, the orf virus obtained from the blood of infected goats was infectious and induced typical symptoms of contagious pustular dermatitis after inoculation of uninfected dairy goats. In summary, our data provide evidence that asymptomatic animals may be carriers of orf virus. Our findings should contribute to elucidating the details underlying the pathogenesis of contagious pustular dermatitis.

Methodologies for Improving HDR Efficiency.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating technology that can be programmed to induce double-strand break (DSB) in the genome wherever needed. After nuclease cleavage, DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. For producing targeted gene knock-in or other specific mutations, DSBs should be repaired by the HDR pathway. While NHEJ can cause various length insertions/deletion mutations (indels), which can lead the targeted gene to lose its function by shifting the open reading frame (ORF). Furthermore, HDR has low efficiency compared with the NHEJ pathway. In order to modify the gene precisely, numerous methods arose by inhibiting NHEJ or enhancing HDR, such as chemical modulation, synchronized expression, and overlapping homology arm. Here we focus on the efficiency and other considerations of these methodologies.