Ethics statement
Animal protocols were approved by the Laboratory Animals Ethics Committee of the Shanghai Animal Disease Control Center.
Phylogenetic analyses
The hemagglutinin (HA) genes of six H3N2 CIV isolates (A/canine/China/Shanghai/0103/2019; A/canine/China/Shanghai/0104/2019; A/canine/China/Shanghai/0105/2019; A/canine/China/Shanghai/018/2019; A/canine/China/Shanghai/019/2019; A/canine/China/Shanghai/0114/2019) from Shanghai in 2019 were used for genetic and phylogenetic analyses (Accession numbers; MK758007.1–MK7580054.1). Sequence data were compiled and edited using Lasergene sequence analysis software (Dnastar Inc., Madison, WI, USA). Multiple sequence alignments were conducted using CLUSTAL W. The maximum likelihood tree for origin analysis was constructed in MEGA 4 using 1,000 bootstrap replications [19, 20].
Virus propagation and electron microscopy
A/canine/Shanghai/0103/2019 (H3N2) was isolated from a stray dog in Shanghai, China. The virus was propagated in 9 day old specific pathogen-free (SPF) embryonated chicken eggs at 37 °C for 48 h and stored at -80 °C. Viral titers were evaluated using the 50% egg-infectious dose (EID50/ml) method and calculated using the Reed–Muench method [21]. For virus isolation using MDCK cells, 1 µg/ml TPCK-treated trypsin was used to support viral growth. Infected cells were incubated under 5% CO2 at 37 °C for 72 h. Supernatants were harvested and further passaged. Then, infection was detected using RT-PCR (Qiagen, Shenzhen). The H3N2 virus (A/canine/Shanghai/0103/2019) was adsorbed onto a carbon parlodion-coated copper grid for 2 min. Excess suspension was removed by blotting with filter paper, and the grid was immediately stained with 1% phosphotungstic acid for 10 min. Excess stain was removed using filter paper, after which samples were examined using a transmission electron microscope (Hitachi, Japan).
Clinical studies and viral challenge
Sixteen beagles (10 weeks old) were obtained from the Experimental Animal Center (Runde Biotechnology Co., Ltd., Shanghai, China). Prior to studies, serum samples were collected from all dogs and subjected to hemagglutination inhibition (HI) assays to ensure that animals had not been exposed to H3N2 CIV. Four dogs were used as controls. For intranasal administration, the 12 remaining dogs were divided into three groups and intranasally inoculated with 1 ml of 104, 105, or 106 EID50 of A/canine/Shanghai/0103/2019. The control group was intranasally inoculated with the same volume of sterile phosphate-buffered saline (PBS). Dogs were housed in separate cages and observed for 10 days after infection.
Nasal swabs were collected, during which time clinical symptoms were monitored. The clinical score of each dog was evaluated using a previously described scoring system [22]. Nasal secretions were collected from left and right nostrils every day until day 10 after inoculation (0–10 days post infection (dpi)) and diluted in 1 ml of PBS plus 1% penicillin and streptomycin. Nasal swabs were used for measuring EID50 values using 9–11 day old embryonic chicken eggs. Two dogs from the control group and two from each experimental group were then humanely euthanized at 5 dpi. Turbinates, tracheas, and lung tissues were collected, and EID50 sample values determined. Briefly, 1 ml of sterile PBS was added per 1 g of collected tissue, which was then ground in a liquid nitrogen homogenizer. Supernatants were collected by centrifugation and inoculated at different dilutions into 9 day old chicken embryos. The two remaining dogs in groups were monitored until day 10.
Serological testing
Blood samples were collected from dogs at 2, 4, 6, 8, and 10 dpi. Approximately 500 µl of serum was collected from each dog and stored at -20 °C until required. One volume of serum was mixed with three volumes of receptor-destroying enzyme (RDE, Denka Seiken Co., Ltd.), incubated for 18 h at 37 °C and then 30 min at 56 °C. Antiserum titers were determined using HI assays. The 1% red blood cells used in this study were collected from SPF cocks and diluted in sterile PBS.
Histopathological examinations using hematoxylin & eosin (H&E) and immunofluorescence (IF) staining
Liver, spleen, lung, trachea, and intestinal tissues collected at 5 dpi were fixed in 10% formalin for > 48 h. Samples were washed overnight, dehydrated in alcohol, and embedded in paraffin. Next, paraffin-embedded tissues were cut into 4–7 μm thick sections and deposited on glass slides. Sections were then mounted and left overnight at 37 °C prior to H&E staining.
IF staining was performed on deparaffinized and rehydrated tissue sections. Briefly, sections were first treated with antigen unmasking solution (Vector Laboratories, California, USA) in a pressure cooker. After blocking in 0.1% Sudan black B for 15 min and 1% bovine serum albumin/PBS at room temperature for 30 min, membranes were incubated with a primary antibody against IAV nucleoprotein (Abcam) at 4 °C overnight. This was followed by incubation with Cy5-conjugated goat anti-mouse IgG (Abcam) for 30 min, after which sections were stained with 4’,6-diamidino-2-phenylindole (Thermo Fisher Scientific). All sections were examined and images captured using a Carl Zeiss LSM780 confocal microscope.
Statistical analyses
Statistical significance was determined using one-way analysis of variance with post hoc Tukey’s multiple-comparison tests using GraphPad Prism (v.7.02) software (GraphPad Software, Inc., La Jolla, CA, USA). P values