Female Kunming mice, 8–9 weeks of age, were purchased from Hunan SJA Laboratory Animal Corporation (Changsha, China). Mice were housed under 12 h light and 12 h dark cycles and controlled temperature (23 ± 3°C). Animals had access to feed and water ad libitum, and were acclimated for 7 d prior to preparation of pregnancy. All animal procedures were performed with the approval of the Ethical Committee of Animal Experiments, Hunan Agricultural University (approval no. 2021.050).

The PRV-YY strain was propagated in a porcine kidney cell line (PK-15) as described previously (12). Female mice (9–10 weeks of age) were housed with proven-fertile males of the same strain. Vaginal plug was checked in the following day morning, and mice carrying plugs were designated as gestation day 0.5 (GD0.5). The timed-pregnant mice were anesthetized with isoflurane inhalation at GD2, and a small incision was introduced on the back to expose the uterine horn. A volume of 50 μL of PRV with either 10⁴ or 10⁵ TCID₅₀ units was injected into each uterine horn using an insulin syringe. Control mice were inoculated with 50 μL of PK-15 cell culture supernatant. After suturing, each animal was placed in an individual cage. Clinical symptoms of mice were monitored three times every day. At GD6, mice were sacrificed, and number of implantation site was recorded. Tissues including thymus, spinal marrow, brain, uterus carrying embryo implantation site, and ovary were collected.

To determine the PRV infectivity, viral nucleic acid was detected in the collected tissues from the PRV-inoculated dam and control mice at GD6. Tissues were homogenized in PBS using a homogenizer (FastPrep-24 Instrument, MP Biomedicals, Irvine, CA, United States). Viral DNA was extracted using a DNA Extraction kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions. Total DNA (500 ng per sample) was subjected to real-time quantitative PCR (RT-qPCR) along with a standard plasmid. The plasmid containing a glycoprotein E (gE) gene, was serially diluted and subjected to RT-qPCR to generate a standard curve using specific primers as described previously (12). The number of PRV copies was determined from the standard curve by converting the corresponding Ct value.

PRV infection was further confirmed in brain and uterus carrying embryo implantation site by in situ hybridization. A DNA probe covering 572 bp of the PRV gD genomic portion was constructed using a PCR-digoxigenin (DIG) Probe Synthesis Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s procedures. The probe was verified by agarose gel electrophoresis. In situ hybridization was performed as described previously (13) with modifications. After deparaffinization and rehydration, tissue sections were pretreated with 10 μg/mL proteinase K at 37°C for 15 min and fixed with 4% cold paraformaldehyde (PFA) for 5 min. Slides were rinsed in distilled water and prehybridized with buffer containing 50% (v/v) deionized formamide in 4 × saline-sodium citrate (SSC). Then sections were incubated with hybridization buffer containing 200 ng/mL DCH-DIG probe at 95°C for 6 min to denature DNA, and cooled on ice for 1 min, then incubated at 42°C overnight in a hybrid furnace. The slides were then serially washed with graded SSC solutions, equilibrated in Buffer I (100 mM Tris, 150 mM NaCl, pH 7.5), blocked with 1% blocking reagent (Boehringer-Mannheim, Indianapolis, IN) in Buffer I, and incubated with anti-DIG-alkaline phosphatase antibody (Roche) at room temperature for 1 h. Sections were washed three times in Buffer I and equilibrated in Buffer III (100 mM Tris, 150 mM NaCl, pH 9.5). Color development was performed using NBT/BCIP (Roche) in Buffer II (100 mM Tris, 100 mM NaCl, 50 mM MgCl₂, pH 9.5) at 4°C overnight. Slides were rinsed in Buffer I, counterstained with a neutral red staining solution and mounted using polyvinylpyrrolidone mounting medium (Beyotime Biotechnology, Haimen, China).

Uterine sections were fixed in 4% PFA for further histological analysis. Tissues were embedded in paraffin, and sectioned. Sections were immersed in xylene to remove paraffin, rehydrated using decreasing grades of ethanol (absolute to 50%) followed by distilled water, and stained with hematoxylin and eosin. Slides were imaged using a microscope. Histology slides were reviewed by a board-certified veterinary pathologist.

Frozen uterine tissues were homogenized in 1 mL TRIzol reagent (Invitrogen, CA, United States). Total RNA was extracted according to the manufacturer’s instructions, and reverse transcribed to cDNA using Vazyme HiScript®III Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China). RNA levels were determined by using Vazyme ChamQTM SYBR®qPCR Master Mix (Vazyme) on a 7,500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, United States). Specific primer sequences were shown in Supplementary Table S1. Levels of PCR products were normalized to the housekeeping gene β-actin using the 2^−ΔΔCT method.

TUNEL assay was performed using a DNA Fragmentation Detection Kit (Beyotime) according to the manufacturer’s instructions. Briefly, the deparaffinized sections were re-hydrated in gradient alcohols, and fixed in 4% PFA followed by PBS wash. Slides were incubated with 20 μg/mL proteinase K (Beyotime) at 37°C for 20 min, and rinsed in PBS twice. TUNEL staining was then performed followed by counterstaining with DAPI (BOSTER, Wuhan, China). The images were collected using a fluorescence microscope.

Frozen uterine samples were homogenized in RIPA buffer (Solarbio, Beijing, China) with protease inhibitor cocktails (Sigma-Aldrich, Saint Louis, MO, United States) and the total proteins were extracted. Concentration of protein was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). Equivalent amounts of total protein (20 μg) were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Immobilon®-PSQ, Bedford, MA, USA). Membranes were blocked in 5% non-fat milk in PBS with Tween 20 for 2 h at room temperature and incubated with indicated antibodies in blocking buffer at 4°C overnight. Primary antibodies included: rabbit anti-Bax antibody (1:3,000, ab182733, Abcam, Cambridge, MA, United States), rabbit anti-Bcl-2 (1:3,000, ab182858, Abcam), and HRP-conjugated mouse anti-beta Actin (1:10,000, KC-5A08, Kangcheng Biotechnology, Shanghai, China). After rinsing with TBST, HRP-conjugated goat anti-rabbit secondary antibody (7074S, Cell Signaling Technology, MA, United States) was incubated on membranes in 5% milk and bands were developed with ECL reagent (KGP1127; Keygen Biotech, Nanjing, China) using ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Hercules, CA, United States). ImageJ was used for densitometry of Western blots.

One-way ANOVA was performed to determine statistically differences followed by Tukey’s multiple comparisons test using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, United States). p value under 0.05 was considered statistically significant.

To investigate whether the PRV would establish infection and cause adverse pregnancy outcomes in pregnant mice, 10⁴ or 10⁵ TCID₅₀ of PRV was inoculated in the pre-implantation mice at GD2 via intrauterine route, respectively. The PRV-inoculated dams exhibited clinical signs of pseudorabies around GD6, and death occurred soon after developing clinical symptoms (Supplementary Figure S1A). Viral nucleic acid was detected in different tissues by RT-qPCR. The copy numbers of PRV genomes were measured in the samples collected from the 10⁵ TCID₅₀ PRV-inoculated dams (n = 3). The results showed that PRV viral loading exhibited discrepancy in different tissues from individual mouse, which was abundant in spinal marrow and brain tissues, and small amounts of PRV genomes were also obtained in thymus, ovary, and uterus carrying embryo implantation site (Figure 1). No viral nucleic acid was detected in tissues from the Mock-treated mice (n = 3). The presence of viral DNA in brain was confirmed using in situ hybridization, but no positive signal was observed in the embryo implantation site of uterus in the PRV-inoculated pregnant mice (Supplementary Figures S1B–E). These findings indicate that PRV can infect mice via intrauterine inoculation.

To determine whether PRV infection cause subfertility in mice, the uteri were examined at GD6 (4 days post-inoculation, dpi) to evaluate the ability of embryo implantation. The results showed that inoculation with 10⁵ TCID₅₀ of PRV significantly decreased mouse embryo implantation (Figure 2A). The mock-treated mice showed an average 13.08 ± 1.93 of implantation sites per mouse (n = 12). Mice infected with 10⁴ TCID₅₀ PRV showed an average 12.00 ± 1.20 implantation sites (n = 8). However, dams infected with 10⁵ TCID₅₀ of PRV carried significantly fewer implantation sites (8.15 ± 4.02, n = 13) than the Mock group (p < 0.001) (Figure 2B). These results demonstrate that intrauterine inoculation of PRV impairs mouse embryo implantation.

H.E. staining was performed to evaluate the histopathological changes in uterus. Compared to the Mock group, the stromal decidua of endometrium was relatively looser in the 10⁴ TCID₅₀ PRV-infected mice with a few inflammatory cells infiltrating (arrow indicate), which was more severe in the 10⁵ TCID₅₀ PRV-infected group (Figure 3A). Nuclei of the majority inflammatory cells were rod-shaped, like neutrophils. We speculated that the decreased ability of embryo implantation was due to the intrauterine inflammatory changes. Therefore, the expression of proinflammatory cytokines including IL-6, IL-1β and TNF-α in uterus was further analyzed by qPCR. The results showed that transcription levels of the three inflammatory factors were significantly increased in 10⁵ TCID₅₀ PRV-inoculated uterus (p < 0.001), and the increase in IL-1β and TNF-α was greater than that of IL-6 (Figure 3B). These results indicate that intrauterine inoculation of PRV induces inflammation and causes endometrial defects in mice.

Apoptosis of the uterine cells was detected by TUNEL staining. In the Mock group, a few apoptotic cells (red fluorescent signal) were found in the stroma, but a medium and large number of apoptotic cells were observed in stroma and endothelium of the endometrium in 10⁴ and 10⁵ TCID₅₀ of PRV-infected animals, respectively; no apoptotic signal was observed in the glandular epithelium (Figure 4A). Bax and Bcl-2 are important pro-apoptotic and anti-apoptotic protein. Western blot assay further showed that the ratio of Bax/Bcl-2 was significantly increased in the 10⁵ TCID₅₀ of PRV-infected group compared with the Mock group (p < 0.01) (Figure 4B).