Objective To evaluate the BALB/c murine infective effects in different concentrations and different aerosol challenge times by Bordetella pertussis.Methods Four experiment groups according to different concentrations and different aerosol challenge times were designed.BALB/c murines were challenged by aerosol way.Group 1: 1010cfu/mL Bordetella pertussis challenge 15 min, group 2: 1010cfu/mL challenge 30 min, group 3: 109cfu/mL challenge 30 min, group 4: 1011cfu/mL challenge 30 min, using the normal saline challenge 30 min as control.At 0d,3d,7d,14d and 21d after challenge, the WBCs of all groups were measured and lung tissues were homogenized to calculate the bordetella pertussis clone in lung.Results After 3 days of challenge, WBCs in all groups were slightly increased.The WBCs of group 1, group 2, group 3 and group 4 were significantly increased after 7 days, with the average numbers of 8.52×109 per/L, 1.74×1010per/L, 1.15×1010per/L and 5×1010per/L, respectively.After 14 days, they were 1.77×1010per/L, 1.67×1010per/L, 1.27×1010per/L and 3.84×1010per/L respectively.WBCs in all groups were dramatically declined after 21 days.The WBC of negative control group had no obvious change during the whole process with the stable number of 3.4~7.0×109per/L.Bordetella pertussis were detected in lung of all experimental groups in each sampling point.The CFU in lung wase at peak at 7d or 14d after challenge, which was obviously decreased at 21d.Conclusion This aerosol challenge method can establish a bordetella pertussis infection mouse model successfully.

Objective:To isolate and identify SARS-CoV-2 epidemic strains and analyze the sequence characteristics of the virus strains following serial passages.Methods:Eleven nasopharyngeal swabs positive for SARS-CoV-2 antigen were collected from December 2022. Quantitative real-time PCR was used to detect SARS-CoV-2 nucleic acid, and positive specimens were inoculated onto Vero cells for virus isolation. The isolated strains were identified by Western blot and indirect immunofluorescence assay. The morphology of the isolated strains was observed using transmission electron microscopy. Nucleic acid was extracted from the isolates and passaged viruses for further sequencing and analysis.Results:All 11 specimens tested positive for SARS-CoV-2 using quantitative real-time PCR. SARS-CoV-2 strains were successfully isolated from seven specimens, and could be adaptively cultured, passaged, and expanded on Vero cells, achieving a peak titer exceeding 10 6.25 50% cell culture infectious dose (CCID 50)/ml. Western blot and indirect immunofluorescence results showed that the isolates could be specifically recognized by monoclonal antibodies and convalescent serum against SARS-CoV-2. Transmission electron microscopy revealed oval-shaped viral particles with diameters of approximately 100 nm. Next-generation sequencing of the viral isolates demonstrated a sequence homology greater than 99.50% with the Wuhan-Hu-1 reference strain (NC_045512) and 99.98% among the seven isolated strains, and all of the isolates belonged to the Omicron BF.7 variant. Sequence analysis after continuous passage and plaque purification of the BJ-NVSI-20230005 isolate showed that compared with passages 1-3, passages 4-6 had one nucleotide site mutation (C→T) in the ORF1ab gene and a deletion of 3 bp in the E gene, which resulted in a change from leucine to phenylalanine and the deletion of valine, respectively. Polymorphisms were observed in the sequences of plaque-purified clones. Conclusions:The seven successfully isolated SARS-CoV-2 strains all belong to the SARS-CoV-2 BF.7 variant, which is consistent with the prevalence trend in mainland China in December 2022.