Journal: Poultry Science
Article Title: Molecular identification and cross-species transmission effects analysis of a novel Orthobunyavirus isolated from Geese in China
doi: 10.1016/j.psj.2025.106223
Figure Lengend Snippet: Isolation and identification of novel Orthobunyavirus CNG01 strain from goose. (A) SPF chicken embryos inoculated with a CNG01-positive homogenate developed systemic hemorrhage and growth retardation by 72 hours post-inoculation. (B) Cytopathic effects (CPE) in Vero cells 72 hours after infection with the CNG01 isolate. (C) RT-PCR detection of the viral NP gene in infected cell culture supernatant. Amplified products were analyzed on a 1.0 % agarose gel (1–5: supernatants; –: negative control). (D) SDS-PAGE analysis of the purified recombinant NP protein. The His-tagged protein was expressed in E. coli and purified by nickel-affinity chromatography, showing a single band of approximately 26 kDa (1–4: washing solutions containing 20, 50, 100 and 250 mM imidazole respectively; 5: elution solution containing 500 mM imidazole). (E) Serum antibody titer measured by ELISA following mouse immunization with the purified NP protein, reaching a titer of 1:512,000. (F) Western blot analysis of NP protein expression in Vero cell lysates 72 hours post-infection. (G) Indirect immunofluorescence assay (IFA) detecting NP protein expression in CNG01-infected Vero cells using mouse anti-NP serum and a FITC-conjugated secondary antibody.
Article Snippet: For IFA, a FITC-labeled goat anti-mouse antibody (Proteintech, SA00013-3, 1:500) was employed.
Techniques: Isolation, Infection, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Agarose Gel Electrophoresis, Negative Control, SDS Page, Purification, Recombinant, Affinity Chromatography, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence