Fenglong Wang

A duck adenovirus type 3 strain, SD2019, was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing the whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells; the cells showed cytopathic effects (CPEs) at 96 h post transfection and the rescued virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFAs). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results show that rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, as well as showing similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3.

A duck adenovirus type 3 strain SD2019 was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells, the cells showed cytopathic effects (CPEs) at 96 hours post transfection and the rescuded virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFA). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results showed that the rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, and showed similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3.

Bovine mastitis is a condition typically induced by various pathogens, with Escherichia coli (E. coli) being a common causative agent known for its propensity to cause persistent infections. In experimental models of bovine mastitis, lipopolysaccharide (LPS), a key component of the E. coli cell wall, is frequently employed as an inducer. The extracellular matrix (ECM) is regulated by MMPs, TIMPs, and the uPA system. They collectively participate in ECM degradation and remodeling and have been identified as promising targets for mastitis treatment. However, investigations into the precise mechanisms underlying E. coli and LPS-induced mastitis, as well as the relationship between bovine mastitis and the MAPK signaling pathway, remain limited. In this study, bovine mammary epithelial cells (BMECs) were treated in vitro with 10⁶ CFU/mL heat-inactivated E. coli, 7.5 µg/mL LPS, or a combination of both. The treatments resulted in varying degrees of activation of the MAPK signaling pathway, specifically ERK1/2, JNK, and P38. BMECs were exposed to MAPK inhibitors (the JNK inhibitor SP600125, the ERK inhibitor PD98059, and the P38 inhibitor SB203580) after treatments with heat-inactivated E. coli (10⁶ CFU/mL), LPS (7.5 µg/mL), or a combination of the two for 6, 12, 24, and 48 h. The mRNA and protein levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, uPA, uPAR, and PAI-1 were assessed using RT-qPCR and Western blot analysis. The findings indicated that heat-inactivated E. coli and LPS stimulated the expression of MAPK mRNAs (ERK1/2, P38, and JNK) in BMECs, along with corresponding increases in the phosphorylated proteins. Furthermore, MAPK inhibitors substantially upregulated the expression of TIMP-1, TIMP-2, and PAI-1. However, no significant changes were observed in the mRNA and protein levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, uPA, or uPAR. In conclusion, heat-inactivated E. coli and LPS can activate the MAPK signaling pathway in BMECs. Inhibiting this signaling pathway can modulate the expression of TIMP-1, TIMP -2, and PAI-1 at both mRNA and protein levels.

Summary: The potato virus Y (PVY) is a plant virus that causes massive crop losses globally, especially in Solanaceae crops. A strain of the plant growth‐promoting rhizobacterium (PGPR), Serratia marcescens ‐S3 was found to inhibit PVY replication in Nicotiana benthamiana . However, there have been no in‐depth studies demonstrating the underlying mechanism. In the current study, we found that ubiquitination of NbHsc70‐2 is an important way for Serratia marcescens ‐S3 to trigger induced systemic resistance (ISR). After the treatment with S. marcescens ‐S3, the protein level of NbHsc70‐2 reduced significantly. Inhibiting of ubiquitination increased the accumulation of NbHsc70‐2 in plants and reduced S. marcescens ‐S3‐mediated resistance to PVY. Furthermore, transgenic engineered Nicotiana benthamiana NbHsc70‐2 ^KO and NbHsc70‐2 ^USM were constructed using CRISPR‐Cas9‐mediated NbHsc70‐2 knock‐out and ubiquitination respectively. S. marcescens ‐S3 significantly reduced the inhibition of NbHsc70‐2 protein accumulation in NbHsc70‐2 ^KO and NbHsc70‐2 ^USM . The virulence of PVY was stronger in NbHsc70‐2 ^USM than the wild‐type plants. These results showed that S. marcescens ‐S3 increases the ubiquitination of NbHsc70‐2 to inhibit the recruitment of molecular chaperone NbHsc70‐2 to reduce its replication and infection of PVY.