Comparative pathogenicity of influenza virus-induced pneumonia mouse model after intranasal and aerosolized intratracheal inoculation | Virology Journal

Mice and viruses

Eight-week-old female wild-type C57BL/6J mice (Vital River Laboratory, Beijing, China) were maintained under specific pathogen-free conditions and had ad libitum access to food and water. The animal experiments were approved by the Animal Ethics Committee of the Academy of Military Medical Sciences (approval number IACUC-DWZX-2023-008).

Mouse-adapted Influenza A/Puerto Rico/8/34 (H1N1) (PR8) [20] was cultured in chicken embryos and the virus-containing allantoic fluid was harvested and stored in aliquots at -80 °C.

Aerosolization of influenza virus solutions

Aerosolization of influenza virus was performed using a MicroSprayer (Huironghe Company, Beijing, China). Anesthetized mice were fixed on the operating table and a laryngoscope was inserted into the deep oral cavity to expose the vocal fold. The MicroSprayer needle was then inserted into the trachea for rapid injection to achieve aerosolization of the influenza virus. The mass mean aerodynamic diameter (MMAD) of the influenza virus aerosol particles was determined using an aerodynamic particle sizer (APS 3321, TSI, USA) with a sampling time of 15 s and a sampling flow rate of 5 L/min. The experiment was repeated three times.

Aerosol distribution of trypan blue and cyanine dye

Mice were anesthetized with 1% pentobarbital sodium solution administered via intraperitoneal injection and immobilized on the operating table. Intranasal inoculation was performed by instilling 50 μL of trypan blue or Cy7.5 through one nostril of the mouse using a pipette gun attached to the tip. Trypan blue and cyanine dye (Cy7.5) were sprayed separately into the lungs of mice using the MicroSprayer for intratracheal aerosol inoculation. The purpose of using trypan blue was to compare the distribution effects of intranasal and aerosolized intratracheal inoculation in the lungs by visual observation, and the difference in fluorescence level was represented by Cy7.5. After trypan blue expulsion, the mice were immediately executed and their lungs were removed. In vivo lung imaging was performed with an IVIS Spectrum small animal imaging system using excitation and emission wavelengths of 770/820 nm after inoculation with the cyanine dye. The isolated lungs were subjected to the same imaging procedure.

Animal experiment protocol

C57BL/6J mice were divided into two groups and vaccinated with influenza viruses at a virus titer of 4.6 PFU, 83.8 PFU, 420 PFU, and 1790 PFU by intranasal or aerosolized intratracheal inoculation, respectively. Lung tissue was collected for histopathological examination, and detection of viral titer and viral load was performed at 1, 3, and 5 days postinfection (dpi). Alveolar lavage fluid was collected for cytokine ELISA assays at 1, 3, and 5 dpi. Lung wet/dry ratio was determined at 1, 3, and 5 dpi and transcriptome analysis was performed at 5 dpi.

Plaque assay and viral load

Mice subjected to the above treatment were euthanized, their lungs were surgically removed and placed in 1 ml DMEM at 1, 3 and 5 dpi. The lung tissue was then homogenized and centrifuged at 12,000 rpm for 10 minutes, which was repeated twice to obtain the supernatant. The supernatant was then filtered through a 0.22 μm filter. MDCK cells were used to determine the virus titer of the supernatant. Total RNA was extracted from 200 μL of supernatant using a PureLink^™ RNA Mini Kit (12183018 A, Thermo Fisher), followed by Q-PCR for viral load detection, with a nucleic acid upload of 100 ng, using primers NP-forward, 5 -GACCRATCCTGTCACCTCTGAC-3; NP-reverse, 5 -GGGCATTYTGGACAAAKCGTCTACG-3; NP probe, TGCAGTCTCGCTCACTGGCACG.

Histopathological test

After intranasal and aerosolized intratracheal infection of C57BL/6J with PR8, mice were euthanized and lungs removed at the indicated time points. Lung tissue was fixed by immersion in 4% formalin for at least one day. The tissues were then embedded in paraffin to form paraffin blocks, which were then sectioned and stained with hematoxylin and eosin. The histopathological score was determined primarily by assessing the extent of alveolar wall thickening, inflammatory cell infiltration, perivascular edema, hemorrhage and bruising using a 4-point grading system [21]. The more severe the above lesion, the higher the histopathological score.

Ratio of wet to dry lungs

The wet-dry ratio of the lungs was used to estimate pulmonary edema. On days 1, 3, and 5 post-exposure, right lung tissue was excised, dried with a clean paper towel, and immediately weighed for wet lung weight (W), followed by incubation of the lungs at 80 °C for 48 hours to acquire dry weight (D ). Finally, the lung W/D ratio was calculated.

ELISA

Bronchoalveolar lavage fluid (BALF) was collected at 1, 3, and 5 dpi. The BALF was obtained by instilling 800 μL of PBS into the trachea of ​​mice followed by three repeated lavages. The collected fluid was then transferred to 1.5 mL EP tubes and centrifuged at 4 °C and 3000g for 10 min. The concentration of cytokines in BALF was determined using a mouse ELISA kit (Solarbio, Beijing, China) according to the instructions provided. The ELISA assay included measurement of IL-6, IL-17 A, MPO, ICAM-1 and IL-1β.