Journal: eBioMedicine

Article Title: Loss of function of Adducin 3 (ADD3) causes abnormal development and impaired barrier function of human and mouse bile duct cells resulting in increased incidence and severity of Biliary Atresia

doi: 10.1016/j.ebiom.2025.106052

Figure Lengend Snippet: Add3 +/− and Add3−/− mouse exhibited a more severe RRV-induced BA phenotypes. (A) Representative pictures of the morphology (Light; Green Fluorescence), histology (H&E) and Sirius Red staining of livers of wild-type ( Add3+/+ ) non-BA neonates (n = 8); RRV-infected wild-type ( Add3+/+ ; n = 16); Add3+/− (n = 17) and Add3−/− (n = 14) BA neonates (Experimental BA) at post-inoculation day 28. “∗” indicates necrotic areas, and was magnified as shown as insets. Arrows indicate portal and periportal fibrosis. Arrowheads indicate portal-to-portal (P-P) bridging fibrosis. (B) Body weight (Mean ± SD) of wild-type ( Add3+/+ ; n = 16); Add3+/− (n = 17) and Add3−/− (n = 14) BA neonates at post-inoculation day 28. Comparative analysis between groups was performed using Kruskal–Wallis test, and p values were shown. Percentages of liver necrosis (C) and fibrosis (D) in wild-type ( Add3+/+ ; n = 16); Add3+/− (n = 17) and Add3−/− (n = 14) BA neonates were shown as Mean ± SD, and p values were shown (Kruskal–Wallis test for quantitative analysis between groups, Dunn's test for multiple comparisons between groups). (E) Serum total bilirubin (TBil), alkaline phosphatase (ALP) and total bile acids levels of wild-type (WT), Add3+/− (C) and Add3−/− (D) neonates with (BA+) and without BA (BA-) phenotypes. Data (Mean ± SD) was analysed between groups using Kruskal–Wallis test, multiple comparisons between groups were performed using Dunn's test, and p values were shown. (WT: BA+, n = 4; BA-, n = 6; Add3+/− : BA+, n = 8; BA-, n = 7; Add3−/− : BA+, n = 4; BA-, n = 5). Abbreviations: li, liver; gb, gall bladder.

Article Snippet: Postnatal day 2 mouse neonates from crossing of Add3+/− mice were injected with RRV (rhesus rotavirus; 20 μl of 1 × 10 6 pfu/ml RRV, MMU 18006, ATCC® VR-1739TM) via peritoneal route.

Techniques: Fluorescence, Staining, Infection

Journal: Journal of Virology

Article Title: Glycoproteins gM and gN are indispensable factors for rhesus macaque rhadinovirus replication and spread but can be reconstituted by KSHV chimeras

doi: 10.1128/jvi.01922-24

Figure Lengend Snippet: Alignments for KSHV and RRV glycoproteins ^a

Article Snippet: The following KSHV-chimeric RRV genomes have been sequenced and deposited into Genbank (NCBI) using the following accession numbers– PQ507925 : KSHVgM-chimeric RRV (KgM-chi.

Techniques:

Journal: Journal of Virology

Article Title: Glycoproteins gM and gN are indispensable factors for rhesus macaque rhadinovirus replication and spread but can be reconstituted by KSHV chimeras

doi: 10.1128/jvi.01922-24

Figure Lengend Snippet: Structural comparisons of gM, gN, and the gM/gN complex from KSHV and RRV. ( A ) Protein alignments of KSHVgM (KgM) (ACY00437.1, from BAC16/JSC-1 KSHV, GQ994935.1) and RRVgM (RgM) (AAD21366.1, in 17577 RRV, AF083501.3), produced using EMBOSS, were color-coded by membrane topology, as determined by DeepTMHMM. To determine amino acid similarity, the BLOSUM50 matrix was applied. N-linked glycosylation motifs were identified and are underlined. Cysteines are marked in pink. ( B ) Similar analytic tools and markings were applied to compare KSHVgN (KgN) (ACY00452.1, in BAC16/JSC-1 KSHV) and RRVgN (RgN) (AAD21379.1, in 17577 RRV). Residues that were identified as probable O-glycosylation sites are marked in orange. ( C ) gM/gN complexes from KSHV, RRV, as well as KSHV/RRV crosses, were generated using AlphaFold and visualized with PDB-3D viewer (gM in green, gN in orange).

Article Snippet: The following KSHV-chimeric RRV genomes have been sequenced and deposited into Genbank (NCBI) using the following accession numbers– PQ507925 : KSHVgM-chimeric RRV (KgM-chi.

Techniques: Produced, Membrane, Glycoproteomics, Generated

Journal: Journal of Virology

Article Title: Glycoproteins gM and gN are indispensable factors for rhesus macaque rhadinovirus replication and spread but can be reconstituted by KSHV chimeras

doi: 10.1128/jvi.01922-24

Figure Lengend Snippet: gM and gN are required for RRV spread, whereas replacement with KSHV gM or gN resulted in replication-competent virus. ( A ) Schematic of BACmid mutagenesis at the RRV gM locus. ( B, C ) qPCR of BAC-transfected 1°RFs, using DNA isolated from cells, presented as viral genome copies log10 quantity(B). ( C ) Western blot using anti-RRV antibodies, recognizing RRV-infected cells. ( D ) Schematic of BACmid mutagenesis at the RRV gN locus. ( E, F ) qPCR of BAC-transfected 1°RFs, using DNA isolated from cells, presented as viral genome copies log10 quantity ( E ). ( F ) Western blot using anti-RRV antibodies, recognizing RRV-infected cells. The black arrows point to perceived mature forms of gB. Viral genome replication was analyzed as described in the Material and Methods. P values of less than 0.0001 (***) were considered extremely significant, whereas P values of less than 0.005 (**) were considered significant.

Article Snippet: The following KSHV-chimeric RRV genomes have been sequenced and deposited into Genbank (NCBI) using the following accession numbers– PQ507925 : KSHVgM-chimeric RRV (KgM-chi.

Techniques: Virus, Mutagenesis, Transfection, Isolation, Western Blot, Infection

Journal: Journal of Virology

Article Title: Glycoproteins gM and gN are indispensable factors for rhesus macaque rhadinovirus replication and spread but can be reconstituted by KSHV chimeras

doi: 10.1128/jvi.01922-24

Figure Lengend Snippet: Glycoprotein-chimeric RRVs express KSHV gM and gN. ( A-B ) At 3–4 weeks post-BAC transfection, RT-PCR was used to examine the gene expression in infected cells, using primers specific for KSHVgM, KSHVgN, vMIP (as a marker for RRV RNA), or GAPDH (as a marker of total RNA). As negative controls, reverse transcriptase (RT) was excluded. As a positive control for the expression of the KSHV genes, RNA was isolated from lytically-reactivated KSHV+ cells (Brk219 cells). ( C ) After constructing, plaque purifying, and titering, single- and double-chimeric RRVs were used to infect 1°RFs (MOI: 1.0), and infected cell lysates were analyzed by western blot for the expression of the KSHV proteins, using newly generated, KSHVgM- and KSHVgN-specific Abs, as well as by RRV-specific Abs. ( D ) Gradient-purified viruses were also evaluated for the incorporation of KSHV proteins by western blot.

Article Snippet: The following KSHV-chimeric RRV genomes have been sequenced and deposited into Genbank (NCBI) using the following accession numbers– PQ507925 : KSHVgM-chimeric RRV (KgM-chi.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Infection, Marker, Reverse Transcription, Positive Control, Expressing, Isolation, Western Blot, Generated, Purification

Journal: Journal of Virology

Article Title: Glycoproteins gM and gN are indispensable factors for rhesus macaque rhadinovirus replication and spread but can be reconstituted by KSHV chimeras

doi: 10.1128/jvi.01922-24

Figure Lengend Snippet: In vitro growth analyses of wild-type and KSHV-chimeric viruses. ( A ) Multi-step growth curve analysis was performed by infecting 1°RFs at an MOI of 0.1, using WT RRV BAC-derived virus as a control. At the time points shown, the cells and supernatants were harvested, freeze-thawed, and sonicated. Infectious virus was measured by plaque assay, with each time point assessment performed in duplicate.

Article Snippet: The following KSHV-chimeric RRV genomes have been sequenced and deposited into Genbank (NCBI) using the following accession numbers– PQ507925 : KSHVgM-chimeric RRV (KgM-chi.

Techniques: In Vitro, Derivative Assay, Virus, Control, Sonication, Plaque Assay