Transgenic C57BL/6J mice expressing hSCARB2 were purchased from the National institutes of Food and Drug Control of China. All mice were housed in a high-efficiency particulate air-filtered individual isolation unit in an Animal Biosafety Level 2-enhanced (ABSL-2+) facility, which complied with the requirements for mouse housing, environment, and comfort as described in the Guide for Laboratory Animals Care issued by the Institute of Medical Biology. The Yunnan Provincial Experimental Animal Management Association and the institutional Experimental Animal Ethics Committee approved the experimental protocols.
Mouse Study Design
A total of 20 transgenic mice (weight: 17.00–20.00 g, 4 weeks old) were randomly divided into the control and CA16 groups. Based our earlier work on nasally infected CA16 tree shrew  and rhesus macaques model , fifteen mice were infected with 104.5 50% cell culture infectious doses (CCID50) of CA16 via the nostrils dropwise. The CA16 virus strain (sub-genotype B) was isolated from a throat swab from an HFMD patient obtained in Guangxi in 2010 (GenBank: JN590244.1) and grown in Vero cells (ATCC, Manassas, VA, USA), which were maintained in Dulbecco's Modifified Eagle Medium (DMEM, HyClone, Logan, UT, USA) supplemented with
10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). While 5 mice used as mock controls were inoculated with the same dosage of phosphate-buffered saline (PBS) via the same route.
After inoculation, the animals were monitored daily for survival and clinical manifestation for 21 days. The onset and duration of all visible changes, such as reduced mobility, limb weakness, paralysis and death, were recorded. Animal feces, and throat swabs were collected daily to detect viral load. Three mice were sacrificed on days 3, 7, 12, 15 and 21 after infection, and the organs or tissues were harvested for viral distribution analysis, histopathology, immunohistochemistry and inflammatory cytokines detection.
Real-Time PCR Test for Viral Load Quantity
Total RNA was extracted from fresh tissue, feces, nasal washes, and blood from the experimental animals using the TRNzol-A + Reagent mini kit (TianGen Biotech, Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The total RNA was eluted in a final volume of 30 µL. For quantification, a single-tube, real-time TaqMan RT-PCR assay was performed using the TaqMan one-step RT-PCR Master Mix in the CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad, Laboratories, Hercules, CA, USA). The experiments were carried out by adding the primer (200 nm), FAM/TAMRA probe (100 nm) (TAKARA Biotechnology Co., Ltd., Dalian, China), and 2 µL of RNA into the TaqMan PCR mater mix, for which the total reaction volume is 20 µL. The following sequences including CA16-specific primers and probe: forward primer, 5’-CTAGTAGTCACAGATTAGGCACTGGTG-3’; reverse primer 5’-CATTGTGATGATGCTGACAAGACC-3’ and the probe 5’FAM-CGTCTAATGCTAGCGACAA-TAMRA-3’ ; The following reaction conditions were applied for all PCR experiments: 5 min at 42 ºC and 10 s at 95 ºC, followed by 40 cycles at 95 ºC for 5 s, and 60 ºC for 30 s. A standard reference curve was established by measuring the serially diluted concentrations of the CA16 RNA standards generated from the in vitro transcription of a DNA gene fragment containing the CA16 p1 gene region.
Histopathological and Immunohistochemical (IHC) Staining
Tissue samples from sacrificed mice were fixed in 10% formaldehyde, dehydrated, embedded, and then cut into 4-µm-thick sections for hematoxylin and eosin (HE) staining assays. For immunohistochemical analysis, the sections were prepared according to the manufacturer’s protocol. Briefly, the slides were deparaffinized, hydrated, antigen-repaired, and then blocked in 4% BSA. CA16 antigen was detected using an anti-enterovirus 71 antibody and cross-reacted with CA 16 antibody (Cat # MAB979, Millipore) prepared by diluting 1:1000 in PBS containing 1% BSA. These slides were washed with PBST and incubated with goat poly-HRP anti-rabbit IgG antibody (Cat # AS040, AB clonal) as a secondary antibody for 35 min at 37 °C. Peroxidase activity was detected with an Enhanced HRP-DAB Chromogenic Substrate Kit (TianGen Biotech, Co., Ltd., Beijing, China). Finally, the slides were examined under a light microscope.
Quantification of Cytokine mRNA
RNA isolations were performed on mouse lung tissue samples with the TRNzol-A + Reagent kit (TianGen Biotech, Co., Ltd., Beijing, China) according to the manufacturer’s protocols. Then, cytokine expression levels were normalized to Beta-actin (β-actin) and are reported as the fold change compared with mock-infected animals. Primer sequences for IL-1β, IL-6, IL-18, TNF-γ and β-actin were published elsewhere. Primer sequences for the remaining genes are as follows: IL-1β: forward, 5’-GAGATGCCTGAGACACCCAAA-3’; reverse, 5’-TGTGCACCAGTTTTCGTTCC-3’; IL-6: forward, 5’-GGAGGCTGTGGATAAACTATTCC-3’; reverse, 5’-CCGGTGTCCACTCAGTGTTTAT-3’; IL-18: forward, 5’-AGAATCAGGCATCCCTCTGC-3’; reverse, 5’-CTTACTGGAGATCCCTGCCG-3’; TNF-γ: forward, 5’ -GTGCTGACGGGACGGTAAA-3’; reverse, 5’ -ACCAGCATCTTTTCCAACCG-3’; Quantitative real-time PCR (qRT-PCR) was performed by using a CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad, Laboratories), and a One Step SYBR PrimeScript™ RT-PCR Kit (TAKARA Biotechnology Co., Ltd.). Each reaction consisted of 1 cycle of 42 ºC for 5 min, 95 ºC for 10 s, followed by 40 cycles of 95 ºC for 5 s and 60 ºC for 30 s. The results of cytokines expression were normalized by β-actin, respectively, and calculated using the 2−ΔΔCT method .
Neutralization Antibody Titer Test
To investigate the dynamic changes of neutralizing antibody response to CA16 in hSCARB2 transgenic mice after infection, serum samples were collected at 3, 7, 12, 14, and 21 days post-infection. CA16-neutralizing antibodies were analyzed using a standard protocol. Briefly, mouse serum was heat-inactivated for 30 min at 56 ºC, then diluted 1:2 in minimum essential medium containing 2% fetal bovine serum (FBS) (Gibco, Life Technologies, Shanghai, China). After that, diluted serum was transferred in triplicate to the first row of one 96-well plate and then diluted two-fold from 1:4 to 1:512. 102.0 hundred CCID50 were combined with the diluted sera in a 96-well, white, opaque-bottom plate and incubated at 35 ºC for 3 h before adding 10,000 Vero cells/well. After incubation, the mixtures were added onto a monolayer of Vero cells and the cells were inspected daily for cytopathic effect (CPE) for up to 4 days. Neutralizing antibody titers were taken to be the highest dilution of serum that inhibited 50% of the viral growth. Neutralization titers were estimated with the Spearman–Karber method and expressed in log2 form (e.g., 4 is a titer of 1:16).
GraphPad Prism 8 (Version 8.0, La Jolla, CA, USA) was used to graph data and to perform statistical analyses. To compare cytokine expression levels between groups, the Mann–Whitney U test was used. Means ± SEMs (standard errors of the mean) were graphed and p < 0.05 was considered to be statistically significant.