Allantois

Recently we demonstrated that NanoLuc (NLuc), a 19-kDa engineered luciferase that possesses 150-fold greater specific activity (that is, light output) than both Renilla and firefly luciferases15 (link), could be stably incorporated into the polymerase subunit (PA) of the WSN virus with no loss of replicative ability in vitro and recapitulates known restrictions due to host range and antiviral treatment in vivo16 (link). Since bioluminescent imaging of viral infections in ferrets has not been reported, we successfully rescued a replication-competent NanoLuc A/California/04/2009 (CA/09-PA NLuc) virus using the eight-plasmid 293T/MDCK co-culture system as described16 (link)19 (link). Viruses were confirmed by sequence analysis and propagated in the allantoic cavity of 10-day-old specific pathogen-free embryonated chicken eggs at 37 °C. Allantoic fluid was harvested, cleared by centrifugation and stored at −80 °C.

Sera were separated by centrifugation at 3000 x g for 10 minutes at 4°C and transferred to sterile Eppendorf tubes. NDV F-specific antibodies were detected using the BioChek Newcastle Disease Virus Antibody Test kit (NDV-F ELISA), according to manufacturer’s instructions. The average sample to positive control ratio (S/P) of four replicates were determined, which was used to calculate the average antibody titre. A S/P ratio ≥ 0.300 is considered as positive, while and antibody titre ≥ 993 is considered as positive. NDV HN-specific antibodies were detected using Hemagglutination inhibition (HI) testing with standard NDV hyperimmune chicken NDV (La Sota) antiserum (Deltamune) by the diagnostic serology laboratory of the University’s Department of Veterinary Tropical Diseases, according to the standard procedures (World Organization for Animal Health (WOAH), 2022 ). HI titres ≥ 4 log2 were considered as positive.
Antisera collected from the adjuvanted treatment group (n=5 birds vaccinated with ND VLPs + Emulsigen^®-P) on day 14 post vaccination were sent to the Animal and Plant Health Agency (APHA)-Weybridge, United Kingdom, for HI analysis and virus neutralisation testing. Six homologous and heterologous NDV isolates from different genotypes were selected from the APHA repository (Supplementary Figure 1), with the amino acid homology being the basis for selection. Although the ND VLP homologous virus (ND isolate turkey/South Africa/N2057/2013) was not available in the APHA repository, an isolate (St. Helena) with more than 99% sequence homology was included. HI testing against each of the six NDV isolates were performed in triplicate, with sera collected from chickens vaccinated with a La Sota-based ND vaccine available for use at APHA included as a positive control. Viruses were used at 4 haemagglutinating units (4 HAU) and HI results interpreted according to standard procedures (World Organization for Animal Health (WOAH), 2022 ). Virus neutralisation tests (VNT) were formed against each of the six antigens (in triplicate), with serum obtained from chickens vaccinated with a La Sota ND vaccine again serving as positive control. Sera were diluted two-fold in Dulbecco’s minimal essential medium (DMEM) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml), 100 TCID₅₀ of selected APMV-1 were added and the samples incubated at 37°C for one hour. Each of the mixtures was subsequently added to DF1 chicken fibroblast cells (ATCC; CRL-12203) for one hour and incubated at 37°C, before the virus/antibody mix was removed by aspiration and replaced with DMEM supplemented with 2.5% [v/v] embryonate fowls’ eggs (EFE) allantoic fluid, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells were incubated for five days at 37°C, whereafter the cytopathic effect was recorded.

Influenza D virus isolate D/bovine/France/5920/2014 (29 (link)) was propagated on specific pathogen-free embryonated chicken eggs (PFIE, INRAE Centre Val de Loire, Nouzilly, France). IDV was titrated by 50% tissue culture infective dose (TCID₅₀) assay using swine testis cells (CRL-1746, ATCC) that were maintained in culture using Dulbecco’s minimum essential medium (DMEM; Gibco) supplemented with 10% heat-inactivated FBS and propagated at 37°C with 5% CO₂. The cells were seeded in in P96-well plates at a density of 10,000 cells/well, and the following day, the culture medium was removed, the cells were washed once with PBS, and the medium was replaced with DMEM supplemented with 2% PS. Virus stocks were 10-fold diluted in the infection medium and were then inoculated on swine testis cells that were incubated for 5 days at 37°C with 5% CO₂. The virus titers were revealed by HA assay using 0.5% solution of chicken red blood cells derived from SPF animals (PFIE, INRAE Centre Val de Loire, Nouzilly, France) and 45 min incubation at +4°C. The titers were determined using the Reed-Muench method. M. bovis strain RM16 (45 (link)) was grown in SP4 medium at 37°C, and the titer was determined by 10-fold dilutions numeration on SP4 agar plates after 5 days of incubation at 37°C. The PCLSs were washed twice with RPMI medium before the infections. Then, the medium was replaced with RPMI supplemented with amphotericin B (2.5 μg/mL; Sigma-Aldrich) and ampicillin (0.3 mg/mL; Sigma-Aldrich). For replication studies, 10³ TCID₅₀ IDV and/or 10³ CFU M. bovis were used (multiplicity of infection [MOI] = 0.001). For innate immunity studies, 10⁶ TCID₅₀ IDV and/or 10⁶ CFU M. bovis were used (MOI = 1). Mock allantoic fluid was inoculated in noninfected and M. bovis conditions. To study the effect of a primary IDV infection on M. bovis superinfection, the PCLSs were infected at 0 h with 10⁶ TCID₅₀ IDV, and 48 h later, 10⁶ CFU of M. bovis were inoculated in superinfected conditions. The supernatants and lung tissues were then collected at 72 and 96 h p.i.