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Japanese encephalitis virus persists in the human reproductive epithelium and porcine reproductive tissues [1]

['Subash Chapagain', 'Vaccine', 'Infectious Disease Organization', 'Vido', 'University Of Saskatchewan', 'Saskatoon', 'Department Of Veterinary Microbiology', 'Western College Of Veterinary Medicine', 'Prince Pal Singh', 'School Of Public Health']

Date: 2022-08 

Japanese encephalitis virus (JEV) is the emerging and geographically expanding flavivirus and the major causative agent of encephalitis in humans in Asia. There are risks of JEV introduction into the Americas given a large population of amplifying hosts—pigs and wild boars, and insect vectors—Culex mosquitoes. There are emerging concerns about vector-free ways of flavivirus transmission, for example sexual and transplacental Zika virus transmissions, which may change flavivirus epidemiology and expand the geographical range to territories with no insect vectors. It is unknown whether JEV has tropism in the female lower reproductive tract and the potential for sexual transmission in humans. While clinical outcomes of transplacental JEV infection are described in humans and pigs, cellular targets and tissue tropism in the upper reproductive tract are also unknown. Here, we studied JEV infection phenotypes and host transcriptional responses in human reproductive epithelial cells. We found that JEV caused persistent infection and cytopathology in the vaginal epithelium, endometrial epithelium, and trophoblast. Human vaginal epithelial cells infected with JEV had altered transcriptional responses associated with inflammation and disruption of epithelial barrier function. Also, using pigs—the native amplifying host for JEV, we confirmed JEV tropism in the female lower and upper reproductive tracts. We discovered that JEV persists in the vaginal mucosa for at least 28 days and pigs shed the virus in vaginal secretions. We also found JEV persistence in the endometrium and placenta with transplacental and fetal infections. Altogether, we discovered that JEV targets the vaginal epithelium and has the potential for sexual transmission in humans. We also contributed to a better understanding of JEV pathogenesis during transplacental infection. Further studies are needed to better understand the interactions of JEV with reproductive tissues, how persistent infection affects female reproductive functions, and the risks for non-vector transmission.

Emerging viruses—newly discovered or with increasing disease incidence—pose a constant threat to public health. The most recent examples of devastating outbreaks of emerging viruses are Ebola virus, new coronaviruses, and Zika virus epidemics. Japanese encephalitis virus (JEV) is the emerging flavivirus related to Zika virus; it is the most important cause of brain infections in Asia that may cause death and severe neurological sequela in patients. Almost half of the world’s population lives in territories where JEV is permanently circulating. Like Zika virus, JEV is transmitted to humans via mosquito bites. However, there are emerging concerns about alternative beyond mosquito-borne ways of flavivirus transmission which may expand the geographical pathogen range to territories with no insect vectors. For example, sexual and transplacental Zika virus transmissions with replication in human vaginal epithelial cells, placenta, and fetuses have been described. Here, we questioned whether JEV also has potential for sexual transmission and studied its tropism in the human female reproductive epithelium—the primary barrier cells of the reproductive system. Also, using pigs—the native amplifying host for JEV, we studied JEV infection in the female lower and upper reproductive tract tissues. We discovered that JEV replicates in different reproductive epithelial cells and tissues of both humans and pigs for a long time, causing molecular and clinical pathology. Thus, further studies are needed to better understand the interactions of JEV with reproductive tissues, how persistent infection affects female reproductive functions, and the risks for non-mosquito transmissions.

Funding: This work was supported by grants to UK from New Frontier in Research Fund (NFRF) # 421586 ( https://www.sshrc-crsh.gc.ca/funding-financement/nfrf-fnfr/exploration/exploration-eng.aspx ) and United States Department of Defense, FY20 PRMRP-Discovery Award # W81XWH-21-1-0014 ( https://cdmrp.army.mil/prmrp/awards/20daawards ). Vaccine and Infectious Disease Organization receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility (InterVac). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Chapagain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Another example of vector-free flavivirus transmission is transplacental Zika virus transmission. Zoonotic flaviviruses were thought to primarily impact human non-reproductive tissues. However, Zika virus replicates in the maternal-fetal interface breaching the placental barrier and infecting the fetal tissues [ 27 – 29 ]. Zika virus crosses the placental barrier to reach the intrauterine cavity with the fetus and replicates in placental trophoblasts. Zika virus infection during all three trimesters of pregnancy may result in fetal infection and congenital abnormalities in newborns; however, severe clinical disease was often attributed to infections in early pregnancy [ 30 ]. While clinical outcomes of transplacental JEV infection are described in humans [ 31 ] and pigs [ 32 ], cellular targets and tissue tropism in the upper reproductive tract are unknown. To better understand JEV pathogenesis during transplacental infection, we studied JEV infection phenotypes in human primary endometrial cells and trophoblast. In pigs, we studied infection in the endometrium, placenta, and fetuses.

There are emerging concerns about alternative, beyond vector-borne ways of flavivirus transmission which may change flavivirus epidemiology and expand the geographical range to territories with no insect vectors. For example, it was thought that JEV transmission occurred only through mosquitoes, but a recent study demonstrated that JEV is transmitted from pig to pig, suggesting vector-free ways for the virus to spread [ 8 ]. Following studies confirmed efficient JEV replication in porcine and human nasal epithelial cells [ 9 , 10 ] and virus oronasal shedding in pigs [ 11 , 12 ] that can enable contact transmission. Another example of vector-free flavivirus transmission is sexual Zika virus transmission. Many sexual transmissions of Zika virus have been described, including male-to-male, male-to-female, female-to-male transmissions [ 13 – 15 ], and fetal infection with congenital Zika syndrome after sexual transmission in mothers [ 16 ]. Rodent and non-human primate models also supported replication in reproductive tissues and sexual transmission of Zika virus [ 17 – 23 ]. In humans, Zika virus has been detected in vaginal secretions [ 24 – 26 ] with replication in vaginal epithelial cells [ 18 ]. It is unknown, however, whether JEV also has tropism in the vaginal epithelium and the potential for sexual transmission in humans. To probe this likelihood, in the present study, we studied JEV infection phenotypes and host transcriptional responses in human primary vaginal epithelial cells. Also, we used pigs—the native amplifying host for JEV and studied infection in vaginal tissues and vaginal virus shedding.

Japanese encephalitis virus (JEV) is a zoonotic flavivirus transmitted by Culex mosquitoes. While efficient vaccines are available [ 1 ], JEV is the major causative agent of encephalitis in humans in the Asia-Pacific region [ 2 , 3 ], with an estimated 68,000 cases reported annually and 15,000 deaths; statistics, however, are most probably underestimated [ 4 ]. Though humans are dead-end hosts of JEV and most human infections are asymptomatic, around 20–30% of clinical infections are fatal, and 30–50% of the survivors develop prolonged or life-long neurological sequelae [ 2 ]. There is no specific cure for the disease; treatment is only supportive to mitigate disease outcomes. The geographic range of emerging JEV keeps expanding from the South of Russia to Australia, including Japan, Eastern China, India, and South-East Asia. For example, the first major outbreak of JEV in Australia with infections in pig herds and human deaths unfolded in February 2022. Also, travel-related severe cases of JEV-induced encephalitis are reported around the globe [ 5 ]. There is a concern that JEV can be introduced into North America given a large population of amplifying hosts—pigs and wild boars; susceptible Culex mosquitoes are also ubiquitous [ 6 , 7 ].

Materials and methods

Ethics statement We followed the Canadian Council on Animal Care guidelines and Animal Use Protocol #20200106 approved by the University of Saskatchewan’s Animal Research Ethics Board and Animal Care and Use Review Office (ACURO) of US Army Medical Research and Development Command. All efforts were made to minimize animal suffering. Pigs were euthanized with an anesthetic overdose followed by exsanguination.

Cells C6/36 cells (ATCC, CRL-1660) were cultured in a Minimum essential medium (MEM; Sigma M4655) supplemented with 10% FBS and 1x Penicillin-streptomycin. VERO E6 cells (ATCC CRL-1586) were cultured in DMEM supplemented with 3% FBS, 1x Penicillin-streptomycin, and 2.67 mM Sodium bicarbonate (Gibco 25080–094). Human vaginal epithelial cells from two donors (ATCC, PCS-480-010 and Lifeline Cell Technology LLC, FC-0083) were cultured in a Vaginal epithelial cell basal medium (ATCC, PCS-480-030) with a Vaginal epithelial cell growth kit (ATCC, PCS-480-040). The commercial supplier confirmed the phenotype by staining cells with epithelium-specific (Pan-CK) and anti-fibroblasts (TE-7) antibodies. Human primary endometrial epithelial cells (Lifeline Cell Technology, FC-0078) were cultured in ReproLife female reproductive epithelial cell culture media with supplements (Lifeline Cell Technology; LL-0068). HTR-8/SVneo trophoblast cells (ATCC CRL-3271) were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI; Gibco 11875119) supplemented with 5% FBS and 1x Penicillin-Streptomycin. VERO E6, human reproductive epithelial cells, and HTR-8/SVneo trophoblast were cultured at +37°C and C6/36 at +28°C in a 5% CO 2 humidified incubator. All cells were mycoplasma free as confirmed by LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich).

Viruses The JEV Nakayama strain (GenBank EF571853) stock was initially produced at the World Reference Center for Emerging Viruses and Arboviruses, the University of Texas Medical Branch at Galveston, and transferred to our facility through the Public Health Agency of Canada. We inoculated VERO E6 cells and harvested media 9 days after inoculation to produce the working stock. Culture media containing JEV was centrifuged (12,000g, 20 min, +4°C); the supernatant was collected, aliquoted, and frozen at -80°C. The virus stock was mycoplasma free as confirmed by LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) We used QIAamp Viral RNA Mini Kit (QIAGEN) according to the manufacturer’s instructions to purify JEV RNA from 140 μl of virus stock, maternal and fetal blood plasma, maternal vaginal and nasal swabs, and the supernatants of different cell cultures. Maternal and fetal tissue samples were dissected and weighed on analytical balances. One ml of TRI Reagent Solution (Thermo Fisher Scientific) was added to 80–100 mg of tissues before homogenization (5 min at 25 Hz) with RNase-free stainless-steel beads and TissueLyser II (QIAGEN). Then, RNA extraction was performed with PhaseMaker tubes (Thermo Fisher Scientific) and PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For JEV RNA quantification, we used the previously described probe-based one-step RT-qPCR assay [33]. All RT-qPCR reactions were conducted on the StepOne Plus platform (Life Technologies, USA) and analyzed using StepOne software version 2.3. The reaction mixture (20 μl) for RT-qPCR (Bioline) consisted of 10 μl 2x SensiFAST Probe One-Step Mix, 0.4 μl RiboSafe RNase Inhibitor, 0.2 μl reverse transcriptase, 1 μl (500 nM) of forward (Universal-JEV-F: 5’-GCCACCCAGGAGGTCCTT-3’) and reverse (Universal-JEV-R: 5’- CCCCAAAACCGCAGGAAT-3’) primers, 0.5 μl (250 nM) probe (Universal-JEV-Probe: 56-FAM-CAAGAGGTG /ZEN/ GACGGCC-3IABkFQ), 1.9 μl nuclease-free water and 4 μl of sample RNA. A reverse transcription step of 10 min at 48°C and an enzyme activation step of 2 min at 95°C were followed by 40 amplification cycles (10 s at 95°C and 20 s at 60°C). RNA from a stock of JEV was used to generate standard curves that had a wide dynamic range (102.5–1012.5 RNA copies/ml) with the high linear correlation R2 = 0.99 between the cycle threshold (Ct) value and template concentration. The standard curve was used to find the detection limit at Ct 40. Assay values were corrected for fluid volumes or tissue weights and upon logarithmical transformation expressed as JEV RNA genome copies per ml or gram. Productive infection in tissues was confirmed with JEV negative-strand-specific RT-PCR: cDNA was synthesized with SuperScript III First-Strand Synthesis System (Invitrogen) using 10 pmole of the JEV-MinusStr forward primer 5-GGTCAGAACCACTACTGACAGT-3. Afterward, cDNA was amplified using the primers Universal-JEV-F and Universal-JEV-R (500 nM of each) and Universal-JEV-Probe (250 nM) described above. An enzyme activation step of 2 min at 95°C was followed by 60 amplification cycles (10 s at 95°C and 20 s at 60°C). In all RNA extraction and PCR assays, we used VERO E6 cell culture media containing JEV as a positive PCR control. As a negative control, we used samples from non-manipulated control animals from our previous studies [34,35]. Strict precautions were taken to prevent PCR contamination. Aerosol-resistant filter pipette tips and disposable gloves were always used. Kit reagent controls were included in every RNA extraction and PCR run.

Detection and quantification of infectious virus We used the endpoint dilution assay in VERO E6 cells to isolate and quantify infectious titers in the JEV stock, blood plasma (maternal and fetal), nasal, and vaginal swabs (maternal) [34–39]. Fluids were serially diluted five-fold in four replicates starting from 1:10 in DMEM media (Thermo Fisher Scientific) supplemented with 5% FBS, a mixture of antibiotics (1,000 IU/ml penicillin and 1 mg/ml streptomycin, Gibco), and 2.25 g/l Sodium Bicarbonate (Thermo Fisher Scientific). Fifty μl of each dilution was added to confluent VERO E6 cells cultured in 96-well plates. After 2 hours of incubation at 37°C, 150 μl of fresh media was added to each well. The cells were incubated for seven days at 37°C. After washing and then drying for at least 4 h, the plates were kept at -20°C for at least 2 hours or until use. Anti-pan flavivirus E protein monoclonal antibodies clone D1-4G2-4-15 (ATCC; HB-112) [40] were used for immunohistochemistry staining [34–37,39] to detect JEV-infected cells. Fifty percent endpoint titers were calculated by the Spearman-Kärber formula and expressed in a decimal logarithm of a 50% infection dose for cell cultures (log 10 TCID 50 ) per ml. Media from mock-inoculated cells were used as negative controls. Maternal blood, nasal swabs, and vaginal swabs which were negative or caused cytotoxic effects on VERO E6 cells, were used to inoculate C6/36 cells for virus isolation. Cells in 96-well plates were inoculated with undiluted or 1:10 diluted blood plasma or swabs in MEM media supplemented with 10% FBS, 1,000 IU/ml penicillin, 1 mg/ml streptomycin, 1x Gentamicin/Amphotericin Solution (Thermo Fisher Scientific), and 2.25 g/l Sodium Bicarbonate. After 12 hours of incubation at 37°C, fluids were removed, replaced with media, and cells were incubated for seven days at 28°C. Afterward, plates were fixed and stained with D1-4G2-4-15 antibodies as described above. To make 10% suspension for titration, maternal tissues were homogenized in media with TissueLyser II (QIAGEN) for 3 min at 20 Hz. Samples were centrifuged at 2,000g for 10 minutes, and after two hours of incubation on C6/36 cells at 37°C, 150 μl of fresh media was added to each well, and cells were incubated for seven days at 28°C. Afterward, plates were fixed and stained with D1-4G2-4-15 antibodies as described above.

In vitro infection phenotypes in human reproductive epithelial cells and trophoblast We used human primary vaginal epithelial cells from two healthy deceased female donors: a 24-years-old African American donor (ATCC, PCS-480-010; Lot Number 80924222; 3rd passage) and an 18-years-old Caucasian donor (Lifeline Cell Technology FC-0083; Lot Number 04033; 3rd passage). Also, we used primary human endometrial epithelial cells from a healthy 13-years-old African American donor (Lifeline Cell Technology FC-0078; Lot Number 09953; 3rd passage). Information on the menstrual phase of human donors was not available, as the samples were de-identified. Cells were free for bacteria, yeast, fungi, mycoplasma, hepatitis B, hepatitis C, HIV-1, and HIV-2, as confirmed by manufacturers with sterility tests and PCR. In JEV studies, three technical replicates were included for cells from each biological donor. Twenty-five thousand vaginal or endometrial epithelial cells were seeded in 96-well plates in appropriate media. The next day, cell monolayers were inoculated with JEV at MOI of 0.1 or 10 in 100 μl of the same media. Plates were incubated at +37°C for 2 hours. Afterward, cells were washed three times with sterile PBS and covered with 200 μl media. Mock-infected cells were included as controls in each plate. Infected plates were incubated (5% CO 2 , +37°C) for 0, 3, 5, and 7 days when supernatants were collected, clarified (2,000 g, 5 min), and frozen (−80°C) for subsequent JEV load quantification. After the supernatant collection, plates with cell monolayer were dried for at least 4 hours and frozen (-80°C). Plates were stained with flavivirus-specific D1-4G2-4-15 antibodies as described above, and infected cells were visualized with a bright-field microscope. The same protocol was used to determine JEV infection kinetics in HTR-8/SVneo trophoblast cells (ATCC CRL-3271).

RNA-seq and bioinformatics Primary vaginal epithelial cells from two human donors were seeded into 24-well plates with 105 cells per well. On the following day, cells in four wells representing technical replicates were inoculated with MOI 10 of JEV prediluted in DMEM media, and four wells were mock-inoculated with virus-free media from VERO E6 cells prediluted in the same way. At 48 hours after inoculation, cells were homogenized in 1 ml of TRI Reagent Solution (Thermo Fisher Scientific), and RNA was extracted according to the manufacturer’s protocol. RNA was assessed on a bioanalyzer and all samples had RNA Integrity Number (RIN) values above 8.0. DNA from samples was removed with TURBO DNA-free Kit (Thermo Fisher Scientific). mRNA with intact poly(A) tails were enriched with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and used for library constructions with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs; New England Biolabs). Libraries were sequenced on the NextSeq as paired-end reads using the NextSeq 500/550 High Output Kit v2.5 (150 cycles) (Illumina). FASTQ files were trimmed for adaptor sequences and filtered for low-quality reads using Trimmomatic. On average, 22.1 million reads per sample were generated. RNA-seq analysis was performed as we previously described [34]. Briefly, a complete transcriptome database was generated from ENSEMBL Homo sapiens GRCh38.p13 (GCA_000001405.28). Sequencing data were mapped and quantified using kallisto [41]. Then counts were analyzed using R BioConductor packages tximport, edgeR and limma. The voom function from the limma package was used for differential expression analysis. Gene set enrichment analysis was performed with camera function in limma using the GMT file (version 7.5.1) containing symbols of gene sets derived from the Gene Ontology Biological Process Ontology of the Gene Set Enrichment Analysis (GSEA) Molecular Signatures Database (MSigDB). The set enrichment results from camera were graphed in Cytoscape using the EnrichmentMap plugin [34,42]. All networks were generated using a Jaccard + Overlap with a cutoff of 0.375 and a Combined Constant of 0.5. Sub-networks were discovered using GLay cluster and annotated using the WordCloud plugin of the top 4 words with a bonus of 8 for word co-occurrence. An accession number for RNA-seq data is PRJNA823367 in NCBI BioProject.

Animal experiment Eight female Landrace-cross pigs were purchased from the university high-health status herd free from porcine reproductive and respiratory virus (PRRSV), porcine parvovirus (PPV), congenital porcine circovirus 2 (PCV2), and porcine circovirus 3 (PCV3), which can cause fetal infection in pigs. Accordingly, maternal and fetal samples were negative for PRRSV, PPV, PCV2, and PCV3 in virus-specific PCR assays [35,43]. Before delivering to containment, six pigs were synchronized and bred with semen from a single donor to reduce biological variability; two pigs (G and H) remained non-pregnant. Insemination was scheduled to ensure that at the time of pig inoculation with JEV, three stages of pregnancy (the duration of pregnancy in pigs is 114 days) were represented: Two pigs at early pregnancy (inoculation at 30 days of pregnancy; pigs A and B), two pigs at mid-pregnancy (54 days; C and D), and two pigs at late pregnancy (86 days; E and F). The pregnancy was confirmed with ultrasound, and all pigs were delivered to Vaccine and Infectious Disease Organization, University of Saskatchewan biosafety level 3 containment facility. Animals were housed in two identical rooms in individual pens with no contact with each other. After seven days of acclimatization in containment, all pigs were sedated and inoculated with 107 TCID 50 of JEV intradermally (ear skin, 1 ml) + intravenous (ear vein, 1ml); this inoculation dose and routes were previously used for JEV inoculation in young piglets [8,44]. Clinical signs, including appetite, activity, and rectal temperature, were recorded before and after JEV inoculation. We collected blood from the jugular vein with BD Vacutainer Plastic Blood Collection EDTA tubes; nasal and vaginal swabs were also collected. Samples were collected before JEV inoculation and at 1–7, 14, 21, and 28 days after virus inoculation. After blood centrifugation (2,000g, 20 min, +4°C), plasma was aliquoted and frozen at -80°C. For nasal and vaginal swabs, swabs were inserted into the top of the nose or vagina and rotated to obtain secretions. Afterward, a swab was placed into a tube containing 500 μl sterile media, the handle was broken one centimeter from the top of the swab, and the tube was stored at -80°C. Pigs were euthanized and sampled 28 days after JEV inoculation. In all pigs, we sampled and froze maternal tonsils, mesenteric lymph nodes, brains, nasal mucosa, and vaginal mucosa with individual sterile instruments. In two non-pregnant pigs, uterine walls with endometrium were sampled and frozen. In six pregnant pigs, uteri with fetuses were removed to sample each fetus (14–15 fetuses per pig) with individual sterile instruments. First, a uterine wall with the placenta was collected from each conceptus (a fetus with fetal membranes) and frozen. Second, umbilical cord blood was aspirated from each fetus with sterile syringes and needles, centrifuged, and plasma was aliquoted and frozen at -80°C. Finally, fetuses were removed, inspected for gross pathology, and whole fetal brains were collected and frozen.

Serology We used an adapted virus-neutralizing assay to quantify JEV-neutralizing antibodies in maternal blood plasma [35,37]. Briefly, 50 μl of JEV (104 TCID 50 /ml) were mixed with equal volumes of two-fold serially diluted plasma (in two replicates) and incubated at +37°C for 1 h before inoculation VERO E6 cells in 96-well plates. After 2 h, 100 μl/well of fresh DMEM supplemented with 1% FBS, 1x Penicillin-Streptomycin and 2.67 mM Sodium Bicarbonate was added. After 7 days, cells were fixed and stained with D1-4G2-4-15 antibodies as described for virus titration. The neutralizing antibody titers were calculated as the highest plasma dilution inhibited JEV infection in 50% of the inoculated wells. We also quantified JEV-specific IgG antibodies in maternal blood plasma with immunoperoxidase monolayer assay (IPMA) [35,37]. Briefly, VERO E6 cells in 96-well cell culture plates were inoculated with 50 μl media containing 104 TCID 50 /ml of JEV and incubated for 2 h (+37°C, 5% CO 2 ). Then 100 μl of the culture medium (DMEM supplemented with 5% FCS, 1x Penicillin/Streptomycin, 2.67 mM Sodium Bicarbonate) was added. After 7 days of incubation at +37°C, 5% CO 2 plates were dried for at least 4 hours and stored at -20°C until use. Plates with cells were thawed, dried, and fixed in 10% buffered formalin for 1 hour, and washed twice with 1x DPBS (pH 7.2). Afterward, fixed cells were incubated with 100% methanol in the presence of 0.3% H 2 O 2 for 10 min. Then plates were washed with DPBS, and two-fold serial dilutions of tested blood plasma were added, followed by 1 hour incubation at +37°C. Plates were washed three times with DPBS containing 0.05% Tween-80 and 50 μl/well of rabbit anti-pig IgG (1:400, Abcam, ab136735) conjugated with horseradish peroxidase were added. After incubation for 1 hour at +37°C and washing, a color reaction was initiated by adding substrate solution: 1 mM 3-amino-9-ethylcarbazole, 5% N,N-dimethylformamide, 50 mM Sodium Acetate (pH 5.0), and 10 mM H 2 O 2 (H 2 O 2 was added just before placing on cells). The reaction was stopped by replacing the substrate with an acetate buffer, and JEV-specific staining was determined by examination with a microscope. The titers were defined as the log reciprocal of the highest serum dilution. Blood plasma samples of mock-inoculated control animals from our previous studies were used as a negative control.

Interferon-alpha quantification To quantify interferon-alpha (IFN-α), maternal and fetal blood plasma samples were diluted 1:2 and tested with Invitrogen Porcine IFN-alpha ELISA Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Immunohistochemistry We used Mouse and Rabbit Specific HRP/DAB IHC Detection Kit—Micro-polymer (Abcam, ab236466) to identify JEV antigen in fetal brains. Fetal brain cryosections of 10 μm were fixed in 10% buffered formalin for 15 min at +4°C. After treatment with 0.3% H 2 O 2 and 1% Triton X-100 for 15 min and protein block for 10 minutes, tissue sections were washed in PBS and incubated with mouse monoclonal antibodies D1-4G2-4-15 (1:10) against flavivirus E protein for 1 hour at +37°C. Afterward, the sections were washed and incubated with mouse specifying reagent, goat anti-rabbit HRP-conjugate, DAB chromogen, and DAB substrate according to the kit’s instructions. Subsequently, tissues were counterstained with hematoxylin and analyzed with a light microscope. For JEV antigen identification in human reproductive epithelial cells and trophoblast, cells were fixed and stained with D1-4G2-4-15 antibodies as described above in the detection and quantification of infectious JEV.

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