Multi-Organ Expression of Viral Entry Receptors in Feline Fetal Tissues

Multi-Organ Expression of Viral Entry Receptors in Feline Fetal Tissues | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multi-Organ Expression of Viral Entry Receptors in Feline Fetal Tissues Anon H Kosaka, Zoe Mia, Nanami X Kato, Asami Oguro-Ando, Akatsuki Saito This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9442451/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Viral receptors play a key role in determining viral organ tropism and host susceptibility to infection. However, distribution of viral receptor expression across feline organs, particularly during the fetal stage, remains poorly understood. In this study, we examined the expression patterns of multiple viral receptor–related genes across fetal cat organs using quantitative polymerase chain reaction. Gene expression levels were quantified as delta cycle threshold values and analyzed using a linear mixed-effects model to account for repeated measurements from the same individual. Initial data visualization showed considerable variation in delta cycle threshold values across different organs and receptors, along with interindividual variability. Statistical analysis using mixed-effects modeling confirmed significant differences in expression patterns among organs and receptors. Notably, several receptors exhibited pronounced organ-specific expression patterns, whereas others showed broader distributions across tissues. In addition, variability among individuals was observed across organ–receptor combinations, highlighting heterogeneity in receptor expression among fetal samples. These findings indicate that viral receptor expression in feline fetal tissues is both organ-specific and subject to interindividual variation. Overall, these results provide a foundational dataset for understanding routes of viral entry and organ tropism in cats and may support future research into feline viral pathogenesis and vertical transmission. Biological sciences/Biological techniques Biological sciences/Computational biology and bioinformatics viral receptor organ tropism gene expression linear mixed-effects model tissue-specific expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cats are widely kept worldwide as important companion animals, and maintaining their health is essential for both animal welfare and public health. Due to their close contact with humans, cats have received particular attention in the context of zoonotic diseases. For instance, severe fever with thrombocytopenia syndrome (SFTS), a tick-borne viral disease, has been reported to be transmitted from infected cats to humans, underscoring the potential role of companion animals as sources of zoonotic infection. Beyond zoonotic concerns, cats are also susceptible to a broad range of viral infections that can lead to severe disease. Despite their clinical significance, the mechanisms by which these viruses establish organ-specific infections, exert pathogenic effects in vivo, and potentially undergo vertical transmission remain poorly understood. Viral receptors play a critical role in determining viral organ tropism and pathways of viral infection. Therefore, understanding their expression patterns across different organs is essential for elucidating viral pathogenesis and potential routes of infection. Although several studies have examined viral infections in cats, systematic comparisons of viral receptor expression across feline organs remain limited. In particular, little is known about receptor expression during the fetal stage, when vertical transmission and developmental susceptibility to infection may occur. In this study, we analyzed the expression of multiple viral receptor–related genes across several fetal organs in cats. By comparing expression patterns among organs, we aimed to define organ-specific receptor expression profiles and provide a foundational dataset for understanding viral organ tropism in felines. The representative feline viruses selected in this study exhibit distinct structural and biological characteristics as well as diverse mechanisms of host cell entry. Below, we summarize the key features of each virus and its associated receptors. Feline coronavirus (a member of the family Coronaviridae ) is an enveloped, positive-sense, single-stranded RNA virus. It is primarily enterotropic and typically causes mild disease [ 1 ]. However, spontaneous mutations in infected cats can lead to the emergence of a macrophage-tropic and often fatal variant known as the feline infectious peritonitis virus ( FIPV ) [ 2 ]. Coronaviruses enter host cells through a two-step process involving spike protein binding to host receptors followed by proteolytic activation. The dendritic cell lectin Cd209 (protein; DC-SIGN) has been reported to facilitate viral entry in certain FIPV serotype II strains [ 3 ]. In the absence of expression data from adult cats, bulk messenger RNA expression data from adult humans indicate relatively high Cd209 expression in adipose tissue, lymph nodes, and the small intestine [ 4 ]. Importantly, FIPV is not primarily defined by organ-specific viral tropism but rather by its strong cellular tropism for macrophages and subsequent systemic dissemination [ 5 ]. Consistent with this, Cd209 is regarded as a cofactor rather than an essential receptor for FIPV entry [ 6 , 7 ]. Transmembrane serine protease 7 ( Tmprss7 , protein; TMPRSS7) belongs to the TMPRSS family and has been implicated in the early stages of coronavirus entry. These host proteases are reported to be important for efficient coronavirus replication [ 8 ] and are involved in multiple stages of the viral life cycle, including cell entry, viral replication, protein maturation, and virion assembly [ 8 , 9 ]. Based on these findings, the present study investigated the expression profiles of viral receptors and Tmprss7 in feline fetal tissues. In cats that succumbed to infection with Dabie bandavirus (formerly Severe fever with thrombocytopenia syndrome virus [SFTSV], family Phenuiviridae ), severe clinical manifestations similar to those observed in fatal human cases of SFTS have been reported. These include high fever, gastrointestinal symptoms, leukopenia, thrombocytopenia, and hepatic and renal dysfunction [ 10 ]. In addition, lesions in lymphoid organs have been consistently observed in both humans and cats [ 10 , 11 ]. Similar to feline coronavirus serotype II, Cd209 has been reported to act as a receptor involved in viral entry into dendritic cells [ 12 ]. Likewise, SFTSV shows broad organ tropism but preferentially infects cell populations such as macrophages and B-cell lineages [ 10 , 11 ]. Although transmission of SFTSV is well documented through tick bites and exposure to blood and body fluids, including human-to-human and cat-to-human transmission [ 10 , 13 ], evidence for vertical transmission remains limited in both humans and cats. In acute feline calicivirus (FCV; family Caliciviridae ) infection, viral antigens are most frequently detected in oral epithelial cells [ 14 ]. FCV primarily infects the epithelial tissues of the upper respiratory tract, oral cavity, conjunctiva, and lungs [ 15 ]. In contrast, in cats with severe systemic FCV disease, the virus can be detected in endothelial cells as well as multiple epithelial and parenchymal cell types, including oral epithelial cells, alveolar epithelial cells, epidermal cells, hepatocytes, and pancreatic acinar cells [ 16 ]. In some strains and disease presentations, FCV can cause systemic infection, with lesions involving multiple organs, including the lungs and intestines [ 16 , 17 ]. During chronic infection, persistent viral shedding in oropharyngeal secretions is observed in a subset of infected cats [ 18 ]. In addition, FCV has been isolated from feces, suggesting that cells in the lower intestinal tract may also support viral replication [ 17 , 18 ]. FCV infection is primarily transmitted horizontally through direct contact, respiratory droplets, or contaminated environments, and there is little evidence supporting established vertical transmission from mother to offspring [ 19 ]. Feline junctional adhesion molecule A ( F11r , protein, also known as JAM-A) is a functional receptor for FCV [ 20 ]. In humans, F11r is expressed not only in epithelial and endothelial cells but also in leukocytes and platelets and is widely distributed in barrier tissues, such as the gastrointestinal tract [ 21 ]. Feline morbillivirus (FeMV; family Paramyxoviridae ) is an emerging infectious agent first identified in 2012 [ 22 ]. Similar to other morbilliviruses, it is suggested to utilize signaling lymphocyte activation molecule family member 1 ( Slamf1 , protein; CD150) as a receptor [ 23 ]. Slamf1 has been proposed to mediate viral entry into immune cells; however, viral replication in target organs, such as the kidney, is likely to involve additional molecules beyond Slamf1 , and Nectin-4 has been suggested as a potential contributing factor [ 24 ]. In humans, Slamf1 is highly expressed in lymphoid tissues, including the thymus and spleen [ 26 , 27 ]. To date, in FeMV-positive cats, histopathological changes such as interstitial inflammatory cell infiltration, as well as degeneration and necrosis of renal tubules, have been observed in the kidney. In addition, FeMV nucleoprotein (N protein) positivity has been detected in renal tubular epithelial cells and mononuclear cells of lymph nodes [ 22 ]. In addition, the urinary tract has been suggested to be an important site for viral replication and persistence [ 27 ]. FeMV infection is primarily transmitted via horizontal routes, with urine-mediated transmission considered one of the most likely pathways. At present, there is no clear evidence supporting vertical transmission [ 28 ]. The absence of detectable viremia further suggests that transplacental infection is unlikely based on current data [ 23 ]. However, because viral RNA has also been detected in the spleen and liver, the possibility of viral replication in other organs cannot be excluded [ 28 , 30 ]. Indeed, in two naturally infected cats, FeMV antigen has been detected in epithelial cells of the urinary bladder, trachea, and bronchioles, as well as in lymphocytes and macrophages of the spleen and mesenteric lymph nodes and in astrocytes and oligodendroglia in the brain [ 23 , 31 ]. Overall, FeMV primarily targets renal tubular epithelial cells and shows tropism for lymphoid and monocytic cell lineages [ 30 ]. The feline panleukopenia virus (FPLV; family Parvovirinae ) attaches to and enters host cells via transferrin receptor 1 ( Tfrc , protein; transferrin receptor 1 TfR1) [ 31 ]. Tfrc is a membrane receptor involved in cellular iron uptake and is widely expressed in humans [ 32 ]. FPLV is highly stable in the environment and highly transmissible, and it causes severe gastrointestinal disease, particularly in young cats [ 33 ]. When infection occurs during pregnancy, vertical transmission may lead to fetal infection and abortion [ 34 ]. FPLV preferentially targets rapidly dividing cells and replicates efficiently in tissues such as the bone marrow, lymphoid organs, and intestinal crypt epithelium [ 35 ]. In particular, proliferating crypt epithelial cells in the intestinal mucosa are major sites of viral replication [ 36 , 37 ]. In addition, infection of lymphoid tissues results in immunosuppression through depleting susceptible cell populations [ 36 , 38 ]. FPLV has also been shown to affect the developing central nervous system, including the cerebellum, during fetal development [ 38 ]. Finally, domestic cat hepadnavirus (DCHBV; family Hepadnaviridae ), first identified in 2018 as an emerging virus [ 39 ], is classified within the genus Orthohepadnavirus . This genus includes the human hepatitis B virus (HBV), which is known to cause hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans [ 40 ]. DCHBV has also been suggested to be associated with hepatitis, liver cirrhosis, hepatocellular carcinoma, and other hepatobiliary disorders in cats [ 41 – 43 ]. Sodium taurocholate cotransporting polypeptide ( Slc10a1 , protein; NTCP) was originally identified as a major entry receptor for HBV [ 44 ]. Our recent study demonstrated that both human and feline Slc10a1 can mediate DCHBV entry [ 45 ]. Furthermore, in humans, perinatal transmission of HBV is a major route of infection [ 46 ], with approximately 90% of infected newborns progressing to chronic infection. HBV can reach the fetus through multiple routes, including infection of placental cells and transfer via peripheral blood mononuclear cells [ 47 ]. Given the close genetic relationship between DCHBV and HBV, a similar mechanism of vertical transmission may also exist in cats. In this study, we quantified and compared the expression of virus receptor–related genes across multiple fetal organs in cats and systematically characterized organ-specific expression patterns. Our findings demonstrate that receptor expression profiles vary substantially among organs and receptor types, with several receptors showing clear organ-specific enrichment. Notably, Slc10a1 exhibited a pronounced liver-specific expression pattern, whereas other receptors, such as Tfrc and F11r , showed were broadly expressed across multiple tissues. These results provide a foundation for understanding viral organ tropism and offer insight into the potential susceptibility of fetal tissues to viral infection. Results Preliminary visualization Preliminary visualization revealed substantial variation in ΔCT distributions across both organs and receptors. Differences in central tendency and dispersion indicated that the data do not follow a single homogeneous distribution. In addition, measurements obtained from the same individual tended to cluster, suggesting the presence of interindividual variability. These observations supported the use of a mixed-effects modeling framework for subsequent analyses. Specifically, central tendency, variability, and the frequency of outliers differed markedly across conditions. Density plots further showed that the location and widths of distribution peaks varied by organ and receptor, reinforcing the lack of a common distributional pattern (Fig. 1 ). Taken together, these findings provide a rationale for employing a mixed-effects model in which individual samples were treated as a random effect. (A) Histogram plots and (B) density plots showing the distribution of delta cycle threshold (ΔCT) values for each receptor gene across different organs. Columns represent organ types (brain, heart, intestine, kidney, liver, lung, spleen, and thymus), and rows represent receptor genes (transferrin receptor 1 [ Tfrc ], signaling lymphocyte activation molecule family member 1 [ Slamf1 ], Cd209 molecule [ CD209 ], junctional adhesion molecule A [ F11r ], solute carrier family 10 member [ Slc10a1 ], and Tmprss7 ). The x-axis indicates delta cycle threshold values, whereas the y-axis represents sample count in (A) and density in (B). Both visualizations demonstrate that expression distributions vary across organ types and receptor genes, with differences in modal values and dispersion. The density plots in (B) provide a smoothed representation of the data and highlight shifts in peak positions across organs and receptors. Density plots of the ΔCT values were generated to visualize expression distributions for each individual within each organ. Even within the same organ, distributions varied across samples, indicating interindividual variability. Moreover, the extent of this variability differed among organs, with certain tissues showing greater dispersion and more distinct separation of distribution peaks. These findings suggest substantial heterogeneity in ΔCT values among individuals in specific organs (Fig. 2 A). Additional density plots overlaying ΔCT distributions for each individual by receptor demonstrated that the degree of inter-sample variability differed across receptors. In particular, Tmprss7 exhibited a broader distribution and more pronounced shifts in peak positions across samples compared with other receptors. In contrast, the distributions for other receptors were relatively similar, indicating lower inter-sample variability (Fig. 2 B). Scatter plots of ΔCT values organized by organ and receptor further showed that expression patterns differed clearly according to both factors (Fig. 3 ). Within the same organ, ΔCT values varied across receptors, indicating receptor-specific expression profiles. At the same time, variation in ΔCT values was observed among individuals even within the same organ–receptor combinations, demonstrating interindividual heterogeneity. The magnitude of this variability differed across both organs and receptors. Notably, Tmprss7 exhibited a broader distribution of ΔCT values compared with other receptors, suggesting greater variability among individuals. In certain organs (such as the heart), differences in the overall ΔCT levels between individuals were also evident. These patterns are more consistent with systematic interindividual differences rather than being driven by an isolated outlier. In contrast, ΔCT distributions for other receptors were more tightly clustered, indicating lower interindividual variability. (A) Scatter plots showing delta cycle threshold (ΔCT) values across organs for each receptor gene. Panels represent receptor genes (transferrin receptor 1 [ TFRC ], signaling lymphocyte activation molecule family member 1 [ SLAMF1 ], CD 209 molecule [ CD209 ], junctional adhesion molecule A [ F11R ], solute carrier family 10 member 1 [ SLC10A1 ], and transmembrane serine protease 7 [ TMPRSS7 ]). The x-axis indicates organ types, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ–receptor combination. (B) Scatter plots showing ΔCT values across receptor genes for each organ. Panels represent organ types. The x-axis indicates receptor genes, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ–receptor combination. These plots demonstrate that expression patterns vary across organs and receptors and reveal interindividual variability within each organ–receptor combination. Mixed-effects modeling Based on these observations, a linear mixed-effects model including sample as a random effect was fitted. Normality of the residuals was assessed using a normal Q–Q plot (Fig. 4 A). The residuals were approximately aligned with the theoretical normal distribution, particularly in the central region, indicating an acceptable overall model fit. Although minor deviations were observed in the tails, no major departures from normality were detected. These findings suggest that the assumption of normally distributed residuals was reasonably satisfied, supporting valid statistical inference from the mixed-effects model. A residuals-versus-fitted plot was used to further evaluate model adequacy (Fig. 4 B). The residuals were randomly distributed around zero across the range of fitted ΔCT values, with no evident systematic patterns such as trends, curvature, or funnel-shaped dispersion. This indicates that the variance of residuals was approximately constant and that no clear violations of model assumptions, such as heteroscedasticity or model misspecification, were observed. Together with the Q–Q plot, these diagnostic results support the appropriateness of the fitted mixed-effects model for subsequent statistical analyses. (A) Normal Q–Q plot of residuals from the linear mixed-effects model. The x-axis represents theoretical quantiles, and the y-axis represents sample quantiles of residuals (defined as observed delta cycle threshold (ΔCT) minus model-predicted ΔCT values). (B) Residuals-versus-fitted values plot from the linear mixed-effects model. The x-axis represents fitted ΔCT values, and the y-axis represents residuals. These plots were used to assess model assumptions, including normality of residuals and homoscedasticity. Post hoc testing This analysis presents the results of post hoc comparisons based on estimated marginal means (EMMs) of ΔCT values derived from the linear mixed-effects model, with organs compared separately for each receptor (Fig. 5 ). The EMMs were calculated after adjusting for interindividual variability by including the sample as a random effect. Each panel corresponds to a single receptor and shows differences in ΔCT values among organs within that receptor. Points represent EMMs of ΔCT, and error bars indicate the corresponding 95% confidence intervals. Letters (a–d) denote statistical groupings based on Sidak-adjusted post hoc tests, with organs sharing the same letter indicating no statistically significant difference. Among the receptors examined, the most notable finding was observed for Slc10a1 , for which the liver exhibited ΔCT values that were significantly different from those of all other organs, indicating a pronounced organ-specific expression pattern. For Tfrc and F11r , significant differences in ΔCT values were detected among multiple organ groups, and both receptors showed relatively high expression levels across organs overall; however, no single organ demonstrated uniquely elevated expression compared with all others. In contrast, signaling lymphocyte activation molecule family member 1 ( Slamf1) showed significantly higher expression in the lung, whereas Cd209 exhibited significantly higher expression in the liver and spleen compared with other organs, highlighting receptor-specific patterns of organ enrichment. For Tmprss7 , although statistically significant differences in ΔCT values among organs were detected, multiple organs were grouped into overlapping statistical categories. Compared with other receptors, organ-dependent differences were more moderate, and overall expression levels tended to be lower across organs. Taken together, these results show that estimated marginal mean ΔCT values vary depending on both receptor and organ. The application of mixed-effects modeling framework enabled the identification of organ-specific expression patterns while appropriately accounting for interindividual variability. Plots showing the estimated marginal means (EMMs) of delta cycle threshold (ΔCT) values across organs for each receptor, derived from the linear mixed-effects model. Panels represent receptor genes. The x-axis indicates organ types, and the y-axis indicates ΔCT values. Points represent EMMs adjusted for interindividual variability by including sample identity as a random effect, and error bars indicate 95% confidence intervals. Letters (a–d) denote the results of post hoc comparisons among organs within each receptor, based on Sidak-adjusted EMMs. Organs sharing the same letter are not significantly different, whereas those with different letters differ significantly. Discussion In this study, we quantified and compared the expression of viral receptor–related genes across multiple fetal organs in cats. Our findings provide the first comprehensive analysis of viral receptor–related gene expression across multiple organs in feline fetuses. To date, systematic comparisons of viral receptor expression across organs, particularly during the fetal stage, have rarely been limited in cats. Our analysis showed that each receptor exhibits distinct organ-specific expression patterns, offering fundamental insights into the mechanisms underlying viral organ tropism and routes of infection. These findings provide new perspectives on how viral infections are established and may help inform the assessment of tissue-specific susceptibility to infection in felines. Our comprehensive analyses showed that Slc10a1 was highly expressed in the liver of feline fetuses (Fig. 5 ). This liver-specific expression pattern is consistent with hepatotropism of DCHBV. In humans, perinatal transmission of HBV represents a major route of infection, with approximately 90% of infected newborns progressing to chronic infection. Furthermore, HBV can reach the fetus through multiple routes, including infection of placental cells and transfer via peripheral blood mononuclear cells [ 47 ]. Given the close genetic relationship between DCHBV and HBV, a similar mechanism of vertical transmission may also exist in cats. Notably, our study demonstrated high Slc10a1 expression in the fetal liver (Fig. 5 ), suggesting that a receptor-mediated mechanism of viral entry may be present during the fetal stage. Although there are currently no definitive reports of vertical transmission of DCHBV, these findings support the possibility of such transmission in cats. Further investigations, including analyses of Slc10a1 expression in adult cat placental tissues of adult cats and elucidation of maternal–fetal transmission mechanisms, are warranted. Cd209 showed enriched expression in the liver and spleen of feline fetuses (Fig. 5 ). Although this expression pattern is consistent with the pathogenesis of FIPV, Cd209 is primarily expressed in specific immune cell populations, such as dendritic cells and macrophages [ 48 – 50 ].Therefore, this pattern may reflect the distribution of these cell types rather than strict organ-level specificity. Accordingly, the observed expression pattern should be interpreted in light of technical limitations, species differences, and developmental stage. Although organ-specific infection and lesion formation are influenced by additional host factors and immune responses, receptor expression in fetal tissues still provides important information regarding potential sites of viral entry. Given the reported 50%–60% mortality rate of SFTS in cats [ 51 ], the expression patterns observed during the fetal stage suggest that SFTSV during pregnancy may lead to fetal death. Taken together, these findings suggest that the establishment of infection and disease outcomes is governed by multiple interacting factors, among which receptor expression is an important determinant of viral susceptibility. Our results showed that Tmprss7 , a serine protease implicated in FIPV entry [ 8 ], exhibited overall low expression and no clear organ specificity in feline fetuses (Fig. 5 ). These findings suggest that Tmprss7 is unlikely to serve as a primary determinant of infection in this context. However, a contributory role in viral entry cannot be excluded, and further functional studies are warranted. F11r was broadly expressed across multiple fetal cat tissues, including the heart, kidney, liver, and thymus (Fig. 5 ). In the fetus, the gastrointestinal system is still developing [ 52 ], and physiological conditions differ substantially from those in postnatal individuals. Accordingly, the expression pattern observed in this study may not directly reflect that of mature animals but may instead reflect developmental-stage–specific regulation. The broad expression of F11r is partially consistent with the tissue distribution of FCV infection, and F11r is known to function as a cell adhesion molecule in epithelial cells [ 53 ]. Although receptor expression alone is insufficient to determine viral tropism, efficient FCV infection may occur in epithelial environments where the receptor is exposed to the external milieu [ 54 ]. Therefore, given that transplacental transmission during viremia cannot be excluded, further investigation of F11r protein localization is warranted. Slamf1 was most highly expressed in the lungs of fetal cats, with additional expression observed in the kidney and thymus (Fig. 5 ). Thus, the lung-dominant expression pattern observed in this study differs from that reported in humans. This discrepancy is likely attributable to the use of fetal tissues and may reflect developmental stage-dependent regulation of gene expression. Fetal organisms are exposed to limited external antigens and exist in a physiological environment distinct from that of postnatal individuals [ 55 ]. Therefore, SLAMF1 expression in fetal tissues may reflect developmental stage–specific immune functions rather than mature activity, and the high expression observed in the lung may represent a preparatory mechanism for postnatal exposure to environmental antigens. Furthermore, FeMV infection is not thought to rely on a single receptor but instead involves a complex entry process mediated by multiple molecules, including Nectin-4 [ 56 ]. Accordingly, Slamf1 expression in fetal tissues is likely associated with developmental processes of the immune system. Tfrc was broadly expressed across multiple fetal feline tissues, with relatively high expression in the spleen and liver (Fig. 5 ). The expression pattern observed in this study is generally consistent with that reported in humans [ 32 ], suggesting that the basic expression profile of Tfrc is conserved across species. The relatively high expression of Tfrc , particularly in the spleen, a key hematopoietic organ during fetal development, is broadly consistent with the known tissue tropism of FPLV. In contrast, Tfrc expression in the fetal brain, which is also susceptible to FPLV infection, was relatively low. This may reflect tissue-specific regulatory mechanisms associated with the unique physiological environment and developmental status of the fetal brain rather than serving as a direct determinant of viral tissue tropism. While we comprehensively analyzed the expression of multiple viral receptor genes in feline fetuses, this study has several limitations. First, because RNA was extracted from whole fetal organs, it was difficult to completely exclude the contribution of circulating blood cells. As a result, some of the observed immune-related gene expression may have been influenced by hematopoietic cells present in the vasculature. Second, the exact gestational age (weeks of pregnancy) of each fetus was not determined, preventing rigorous comparison of receptor expression across defined developmental stages and limiting interpretations of stage-dependent variation. Third, due to the small size of fetal organs, it was not possible to separate epithelial, stromal, and endothelial compartments during tissue sampling. Consequently, the expression data represent average signals across whole organs, and the assessment of microanatomical localization was not feasible. Nevertheless, this study provides the first comprehensive analysis of multiple viral receptor genes in feline fetuses, thereby contributing to a better understanding of the potential mechanisms underlying vertical transmission of viral infections. Regarding future directions, it will be necessary to precisely determine fetal age and conduct longitudinal analyses to clarify developmental stage-dependent changes in receptor expression. It will also be important to investigate whether single-nucleotide polymorphisms in receptor genes are associated with viral susceptibility or organ specificity. Since FPLV is known to cause transplacental infection, assessing receptor expression in placental and maternal tissues may help elucidate the molecular mechanisms underlying vertical transmission. Moreover, this study was limited to RNA-level analysis, and whether the corresponding proteins are actually expressed remains unknown—representing an important limitation. Even when receptor messenger RNA is detected, posttranslational regulation or translational suppression may prevent protein expression [ 58 , 59 ]. Therefore, future studies should include verification at the protein level, such as immunohistochemistry or proteomic approaches, to confirm receptor expression and tissue distribution. In conclusion, this study quantitatively characterized the expression of viral receptor–related genes across multiple fetal organs in cats and demonstrated that receptor expression profiles vary markedly across both organs and receptor types. Distinct organ-specific enrichment was observed for several receptors, including liver-specific expression of Slc10a1 , whereas other receptors, such as Tfrc and F11r , showed broader tissue distribution. These findings support our initial hypothesis that receptor expression is organ-dependent and provide a framework for understanding viral organ tropism. While receptor expression alone is insufficient to determine infection outcomes, our results suggest that it is an important factor contributing to viral susceptibility, particularly in the context of DCHBV. Future studies incorporating developmental stage, cellular resolution, and protein-level validation will be essential to further elucidate the mechanisms underlying viral infection and pathogenesis in felines. Materials and methods Ethical approval All procedures involving the collection of feline samples were approved by the Animal Care and Use Committee of the University of Miyazaki (Approval No. 2023-012) and were conducted in accordance with the University of Miyazaki experimentation regulations and relevant institutional guidelines. Animal Research: Reporting of In Vivo Experiments statement This study is reported in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines. No live animals were used specifically for this study. The fetal tissues analyzed were obtained from fetuses that were already dead at the time of collection from a surgically removed uterus. Therefore, no euthanasia or experimental animal procedure was performed for this study. Organ collection Organ collection was performed as follows. The uterus was obtained from a cat that underwent ovariohysterectomy for medical reasons at a university-affiliated veterinary hospital in Japan and was kept on ice until use. The fetuses were already dead at the time the uterus was collected. Therefore, no animals were euthanized for the purposes of this study, and no euthanasia method or anesthetic agent for fetal sacrifice was applicable. The uterus was dissected to retrieve the fetuses. From each fetus, tissue samples of the lung, intestine, spleen, kidney, heart, brain, liver, and thymus were carefully excised using sterile scissors and forceps. Each tissue sample was immediately placed into a 1.5 mL microcentrifuge tube containing RNAlater Stabilization Solution (Thermo Fisher Scientific, catalog no. AM7020) and stored at −30°C until RNA extraction. No predetermined gestational age was available for the fetuses, and the age of the mother cats was also unknown. RNA extraction Tissues were initially disrupted using a Biomasher (Nippi, Inc., catalog no. 893064). The homogenized tissues were then lysed in Buffer RLT supplemented with 2-mercaptoethanol (Bio-Rad, Hercules, CA, USA, Catalog no. 1610710). RNA was subsequently purified according to the RNeasy Mini Kit protocol (Qiagen, catalog no. 74104). RNA was eluted with 90 μL of RNase-free water, and RNA concentrations were measured using a NanoDrop Eight spectrophotometer (Thermo Fisher Scientific, Inc., catalog no. NDE-GL). Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction was performed using the One Step TB Green® PrimeScript™ PLUS Real-Time Polymerase Chain Reaction Kit (Takara Bio Inc., catalog no. RR096A) on a QuantStudio5 Real-Time Polymerase Chain Reaction System (Thermo Fisher Scientific). Primers listed in Supplemental Table 1 were used in this study. Gene expression levels were quantified using the comparative ΔCt method. ΔCt values were calculated as Ct(target gene) – Ct ( ribosomal protein S7 ( Rps7 )), where Rps7 was used as the reference housekeeping gene [59]. All qRT-PCR reactions were performed in technical triplicate. For each sample, triplicate Ct values were inspected for consistency. When all three replicates produced valid Ct values, the mean Ct was used for subsequent analysis. If one replicate showed clear deviation from the others (e.g., due to technical error), it was excluded, and the mean of the remaining replicates was used. Samples with insufficient valid replicates were treated as missing data. Undetermined Ct values were treated as missing values and were not imputed. These missing observations were retained as NA in downstream analyses. Potential outliers were initially identified based on technical inconsistency and statistical criteria (see below). However, outliers were not removed from the primary analysis unless there was clear evidence of technical error. Sensitivity analyses were performed with selected outliers removed to assess the robustness of the results. Statistical analysis To account for the hierarchical structure of the data, a linear mixed-effects model was fitted using the lme4 package in R. To account for the hierarchical structure of the data, a linear mixed-effects model was fitted using the lme4 package in R. Organ and receptor were treated as fixed effects, and their interaction term (organ × receptor) was included in the model. Sample identity (individual cat) was included as a random intercept to account for repeated measurements obtained from the same individual. Model comparison was performed to assess the contribution of the interaction term by comparing models with and without the interaction using likelihood ratio tests. Post hoc comparisons among organs within each receptor were conducted using estimated marginal means implemented in the emmeans package. Multiple comparisons were adjusted using Sidák correction within each receptor. Model assumptions were evaluated using residual-versus-fitted plots to assess homoscedasticity and Q–Q plots to assess normality of residuals. A p-value of 0.05 or less was considered statistically significant. All analyses were conducted using R version 4.5.2 (2025-10-31). Declarations Acknowledgments The authors thank Ms. Tomoko Nishiuchi, Ms. Miki Kawano, Ms. Natsumi Matsubara, and the staff of CADIC, University of Miyazaki, for their assistance. Author information Contributions AH.K., and A.S. designed the experiments. AH.K., and NX.K performed the experiments. AH.K., NX.K, Z.M, A.O-A, and A.S. analyzed the results. M.S., Z.M., A.O-A., and A.S. wrote and edited the manuscript. All authors have read and approved the manuscript. Data availability Source data are available on request. Funding This work was supported by grants from the Japan Agency for Medical Research and Development (AMED) Research Program on HIV/AIDS JP26fk0410075, JP26fk0410080, JP25fk0410056, and JP25fk0410058 (to A.S.); the AMED Program for Accelerating Medical Research JP256f0137007j0001 (to A.S.); the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) JP24K09227 (to A.S.); the JSPS KAKENHI Grant-in-Aid for Scientific Research (B) JP22H02500 (to A.S.) and JP21H02361 (to A.S.); the JSPS Bilateral Program JPJSBP120245706 (to A.S.); the JSPS Fund for the Promotion of Joint International Research (International Leading Research) JP23K20041 (to A.S.); and the G-7 Grants (2025 and 2026) (to A.S.). This study was supported by the Frontier Science Research Center, University of Miyazaki. Ethics declarations All procedures involving the collection of feline samples were approved by the Animal Care and Use Committee of the University of Miyazaki (approval No. 2023-012) and conducted in accordance with the University of Miyazaki Experimentation regulations and relevant institutional guidelines. Competing interests The authors declare no competing interests. Corresponding author Asami Oguro-Ando and Akatsuki Saito References Tasker, S. et al. Feline Infectious Peritonitis: European Advisory Board on Cat Diseases Guidelines. Viruses 15 , (2023). Vennema, H., Poland, A., Foley, J. & Pedersen, N. C. Feline Infectious Peritonitis Viruses Arise by Mutation from Endemic Feline Enteric Coronaviruses. Virology 243 , 150–157 (1998). Regan, A. D. & Whittaker, G. R. Utilization of DC-SIGN for Entry of Feline Coronaviruses into Host Cells. J Virol 82 , 11992–11996 (2008). Li, J. et al. 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Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells. J. Virol. 75 , 3896–3902 (2001). Guo, Q., Qian, C., Wang, X. & Qian, Z.-M. Transferrin receptors. Exp Mol Med 57 , 724–732 (2025). Kabir, A. et al. Epidemiology and molecular characterization of Feline panleukopenia virus from suspected domestic cats in selected Bangladesh regions. PLoS One 18 , e0282559 (2023). Truyen, U. et al. Feline Panleukopenia: ABCD Guidelines on Prevention and Management. Journal of Feline Medicine and Surgery 11 , 538–546 (2009). Parrish, C. R. 3 Pathogenesis of feline panleukopenia virus and canine parvovirus. Baillière’s Clinical Haematology 8 , 57–71 (1995). Carlson, J. H. & Scott, F. W. Feline Panleukopenia: II. The Relationship of Intestinal Mucosal Cell Proliferation Rates to Viral Infection and Development of Lesions. Vet Pathol 14 , 173–181 (1977). Stuetzer, B. & Hartmann, K. Feline parvovirus infection and associated diseases. The Veterinary Journal 201 , 150–155 (2014). Pfankuche, V. M. et al. Neuronal Vacuolization in Feline Panleukopenia Virus Infection. Vet Pathol 55 , 294–297 (2018). Aghazadeh, M. et al. A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia. Viruses 10 , (2018). Revill, P. A. et al. A global scientific strategy to cure hepatitis B. Lancet Gastroenterol Hepatol 4 , 545–558 (2019). Dosaka, H. et al. Distribution of domestic cat hepatitis B virus in cholangiocarcinoma and non-neoplastic liver tissue. J Vet Med Sci https://doi.org/10.1292/jvms.25-0372 (2026) doi:10.1292/jvms.25-0372. Pesavento, P. A. et al. A Novel Hepadnavirus is Associated with Chronic Hepatitis and Hepatocellular Carcinoma in Cats. Viruses 11 , (2019). Piewbang, C. et al. Domestic cat hepadnavirus associated with hepatopathy in cats: A retrospective study. J Vet Intern Med 36 , 1648–1659 (2022). Tan, X., Xiang, Y., Shi, J., Chen, L. & Yu, D. Targeting NTCP for liver disease treatment: A promising strategy. J Pharm Anal 14 , 100979 (2024). Shofa, M., Ohkawa, A., Kaneko, Y. & Saito, A. Conserved use of the sodium/bile acid cotransporter (NTCP) as an entry receptor by hepatitis B virus and domestic cat hepadnavirus. Antiviral Research 217 , 105695 (2023). Hu, Y. & Yu, H. Prevention strategies of mother-to-child transmission of hepatitis B virus (HBV) infection. Pediatric Investigation 4 , 133–137 (2020). Sirilert, S. et al. Possible Association between Genetic Diversity of Hepatitis B Virus and Its Effect on the Detection Rate of Hepatitis B Virus DNA in the Placenta and Fetus. Viruses 15 , (2023). Soilleux, E. J. et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol. 71 , 445–457 (2002). Švajger, U., Anderluh, M., Jeras, M. & Obermajer, N. C-type lectin DC-SIGN: An adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cellular Signalling 22 , 1397–1405 (2010). Yamada, R. et al. Expression of macrophage/dendritic cell–related molecules in lymph node sinus macrophages. Microbiology and Immunology 67 , 490–500 (2023). Matsuu, A. et al. Natural severe fever with thrombocytopenia syndrome virus infection in domestic cats in Japan. Veterinary Microbiology 236 , 108346 (2019). Indrio, F. et al. Development of the Gastrointestinal Tract in Newborns as a Challenge for an Appropriate Nutrition: A Narrative Review. Nutrients 14 , (2022). Wang, J. & Chen, X. Junctional Adhesion Molecules: Potential Proteins in Atherosclerosis. Front. Cardiovasc. Med. 9 , (2022). Asif, S., Yingkun, D. & Meng, C. Unlocking the secrets of Feline calicivirus: advances in structural and nonstructural proteins and its role as a key model for other Caliciviruses. Virol J 22 , 152 (2025). Rackaityte, E. & Halkias, J. Mechanisms of Fetal T Cell Tolerance and Immune Regulation. Front. Immunol. 11 , (2020). Sato, H., Yoneda, M., Honda, T. & Kai, C. Morbillivirus Receptors and Tropism: Multiple Pathways for Infection. Front. Microbiol. 3 , (2012). Buccitelli, C. & Selbach, M. mRNAs, proteins and the emerging principles of gene expression control. Nat Rev Genet 21 , 630–644 (2020). Naeli, P., Winter, T., Hackett, A. P., Alboushi, L. & Jafarnejad, S. M. The intricate balance between microRNA-induced mRNA decay and translational repression. The FEBS Journal 290 , 2508–2524 (2023). Penning, L. C. et al. A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues. Vet Immunol Immunopathol 120 , 212–222 (2007). Additional Declarations No competing interests reported. Supplementary Files 260417SupplementaryTable1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 09 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 25 Apr, 2026 Editor assigned by journal 25 Apr, 2026 Editor invited by journal 24 Apr, 2026 Submission checks completed at journal 21 Apr, 2026 First submitted to journal 21 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9442451","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":637494739,"identity":"8c77e86e-011d-470c-b822-5e044aee87de","order_by":0,"name":"Anon H Kosaka","email":"","orcid":"","institution":"University of Miyazaki","correspondingAuthor":false,"prefix":"","firstName":"Anon","middleName":"H","lastName":"Kosaka","suffix":""},{"id":637494740,"identity":"c0b38989-96d8-48e9-8df8-b98e731153b2","order_by":1,"name":"Zoe Mia","email":"","orcid":"","institution":"University of Exeter","correspondingAuthor":false,"prefix":"","firstName":"Zoe","middleName":"","lastName":"Mia","suffix":""},{"id":637494741,"identity":"1de97e61-7ff6-422d-86e7-f1e3e12542ae","order_by":2,"name":"Nanami X Kato","email":"","orcid":"","institution":"University of Miyazaki","correspondingAuthor":false,"prefix":"","firstName":"Nanami","middleName":"X","lastName":"Kato","suffix":""},{"id":637494742,"identity":"85e4b8fc-777b-4e33-bfe0-dcd4beb170b3","order_by":3,"name":"Asami Oguro-Ando","email":"","orcid":"","institution":"University of Exeter","correspondingAuthor":false,"prefix":"","firstName":"Asami","middleName":"","lastName":"Oguro-Ando","suffix":""},{"id":637494743,"identity":"e87ecbef-bac9-4b6e-bf7a-0c561e4ade56","order_by":4,"name":"Akatsuki Saito","email":"data:image/png;base64,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","orcid":"","institution":"University of Miyazaki","correspondingAuthor":true,"prefix":"","firstName":"Akatsuki","middleName":"","lastName":"Saito","suffix":""}],"badges":[],"createdAt":"2026-04-16 23:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9442451/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9442451/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109112017,"identity":"05d84543-919e-4c2d-9aeb-26c793d221c7","added_by":"auto","created_at":"2026-05-12 15:47:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1036866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of receptor gene expression across organs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Histogram plots and (B) density plots showing the distribution of delta cycle threshold (ΔCT) values for each receptor gene across different organs. Columns represent organ types (brain, heart, intestine, kidney, liver, lung, spleen, and thymus), and rows represent receptor genes (transferrin receptor 1 [\u003cem\u003eTfrc\u003c/em\u003e],\u003cem\u003e signaling lymphocyte activation molecule family member 1 \u003c/em\u003e[\u003cem\u003eSlamf1\u003c/em\u003e],\u003cem\u003e Cd209 molecule \u003c/em\u003e[\u003cem\u003eCD209\u003c/em\u003e],\u003cem\u003e junctional adhesion molecule A \u003c/em\u003e[\u003cem\u003eF11r\u003c/em\u003e],\u003cem\u003esolute carrier family 10 member \u003c/em\u003e[\u003cem\u003eSlc10a1\u003c/em\u003e], and \u003cem\u003eTmprss7\u003c/em\u003e). The x-axis indicates delta cycle threshold values, whereas the y-axis represents sample count in (A) and density in (B). Both visualizations demonstrate that expression distributions vary across organ types and receptor genes, with differences in modal values and dispersion. The density plots in (B) provide a smoothed representation of the data and highlight shifts in peak positions across organs and receptors.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/8a42ac0301348552356ca149.png"},{"id":109204616,"identity":"c7683fb1-05d8-44b8-a754-a4a1d0a3f488","added_by":"auto","created_at":"2026-05-13 15:01:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1004546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInterindividual variability in receptor gene expression across organs and receptors \u003c/strong\u003e(A) Density plots showing the distribution of delta cycle threshold (ΔCT) values for each individual sample across different organs. Each colored line represents a single sample, and panels correspond to organ types. The x-axis indicates ΔCT values, and the y-axis represents density. (B) Density plots showing the distribution of ΔCT values for each individual sample across receptor genes. Each colored line represents a single sample, and panels correspond to receptor genes (transmembrane serine protease 7 [\u003cem\u003eTMPRSS7\u003c/em\u003e] and others). The x-axis indicates ΔCT values, and the y-axis represents density. These visualizations highlight interindividual variability in expression patterns. In (A), variability within the same organ differs across tissues, with some organs showing greater dispersion and more distinct separation of distribution peaks. (B) The degree of variability varies among receptors, with Tmprss7 exhibiting broader distributions and more pronounced shifts in peak positions across samples compared with other receptors.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/afb94e0b9268f0ca71992e30.png"},{"id":109204557,"identity":"27d21307-d955-469b-be79-d3bd1a248a51","added_by":"auto","created_at":"2026-05-13 15:01:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":735173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of ΔCT values across organs and receptors at the individual level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scatter plots showing delta cycle threshold (ΔCT) values across organs for each receptor gene. Panels represent receptor genes (transferrin receptor 1 [\u003cem\u003eTFRC\u003c/em\u003e], signaling lymphocyte activation molecule family member 1 [\u003cem\u003eSLAMF1\u003c/em\u003e], CD 209 molecule [\u003cem\u003eCD209\u003c/em\u003e], junctional adhesion molecule A [\u003cem\u003eF11R\u003c/em\u003e], solute carrier family 10 member 1 [\u003cem\u003eSLC10A1\u003c/em\u003e], and transmembrane serine protease 7 [\u003cem\u003eTMPRSS7\u003c/em\u003e]). The x-axis indicates organ types, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ–receptor combination. (B) Scatter plots showing ΔCT values across receptor genes for each organ. Panels represent organ types. The x-axis indicates receptor genes, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ–receptor combination. These plots demonstrate that expression patterns vary across organs and receptors and reveal interindividual variability within each organ–receptor combination.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/fde310b287b39156c5d6ae41.png"},{"id":109205102,"identity":"a6aae144-88e6-431e-9e79-4ccd898a19e1","added_by":"auto","created_at":"2026-05-13 15:03:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":570149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagnostic plots for evaluating the linear mixed-effects model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Normal Q–Q plot of residuals from the linear mixed-effects model. The x-axis represents theoretical quantiles, and the y-axis represents sample quantiles of residuals (defined as observed delta cycle threshold (ΔCT) minus model-predicted ΔCT values). (B) Residuals-versus-fitted values plot from the linear mixed-effects model. The x-axis represents fitted ΔCT values, and the y-axis represents residuals. These plots were used to assess model assumptions, including normality of residuals and homoscedasticity.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/0229e2138af488d73b838683.png"},{"id":109112014,"identity":"4f80cb3c-8ab3-468e-b80a-1eea4241c713","added_by":"auto","created_at":"2026-05-12 15:47:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":301276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstimated marginal means of delta cycle threshold (ΔCT) values across organs for each receptor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlots showing the estimated marginal means (EMMs) of delta cycle threshold (ΔCT) values across organs for each receptor, derived from the linear mixed-effects model. Panels represent receptor genes. The x-axis indicates organ types, and the y-axis indicates ΔCT values. Points represent EMMs adjusted for interindividual variability by including sample identity as a random effect, and error bars indicate 95% confidence intervals. Letters (a–d) denote the results of post hoc comparisons among organs within each receptor, based on Sidak-adjusted EMMs. Organs sharing the same letter are not significantly different, whereas those with different letters differ significantly\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/3550733140ffe79d274b814a.png"},{"id":109207342,"identity":"f966e27e-b0a6-4e90-9488-79c393a0602c","added_by":"auto","created_at":"2026-05-13 15:19:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3247850,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/3e596f41-411f-484b-816b-7bae29948ea7.pdf"},{"id":109204743,"identity":"0a65113e-8ba1-4914-a199-25878284dbc3","added_by":"auto","created_at":"2026-05-13 15:02:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":68271,"visible":true,"origin":"","legend":"","description":"","filename":"260417SupplementaryTable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9442451/v1/a080f9ac809eef55dc833958.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multi-Organ Expression of Viral Entry Receptors in Feline Fetal Tissues","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCats are widely kept worldwide as important companion animals, and maintaining their health is essential for both animal welfare and public health. Due to their close contact with humans, cats have received particular attention in the context of zoonotic diseases. For instance, severe fever with thrombocytopenia syndrome (SFTS), a tick-borne viral disease, has been reported to be transmitted from infected cats to humans, underscoring the potential role of companion animals as sources of zoonotic infection. Beyond zoonotic concerns, cats are also susceptible to a broad range of viral infections that can lead to severe disease. Despite their clinical significance, the mechanisms by which these viruses establish organ-specific infections, exert pathogenic effects in vivo, and potentially undergo vertical transmission remain poorly understood.\u003c/p\u003e \u003cp\u003eViral receptors play a critical role in determining viral organ tropism and pathways of viral infection. Therefore, understanding their expression patterns across different organs is essential for elucidating viral pathogenesis and potential routes of infection. Although several studies have examined viral infections in cats, systematic comparisons of viral receptor expression across feline organs remain limited. In particular, little is known about receptor expression during the fetal stage, when vertical transmission and developmental susceptibility to infection may occur. In this study, we analyzed the expression of multiple viral receptor\u0026ndash;related genes across several fetal organs in cats. By comparing expression patterns among organs, we aimed to define organ-specific receptor expression profiles and provide a foundational dataset for understanding viral organ tropism in felines.\u003c/p\u003e \u003cp\u003eThe representative feline viruses selected in this study exhibit distinct structural and biological characteristics as well as diverse mechanisms of host cell entry. Below, we summarize the key features of each virus and its associated receptors.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFeline coronavirus\u003c/em\u003e (a member of the family \u003cem\u003eCoronaviridae\u003c/em\u003e) is an enveloped, positive-sense, single-stranded RNA virus. It is primarily enterotropic and typically causes mild disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, spontaneous mutations in infected cats can lead to the emergence of a macrophage-tropic and often fatal variant known as the \u003cem\u003efeline infectious peritonitis virus\u003c/em\u003e (\u003cem\u003eFIPV\u003c/em\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Coronaviruses enter host cells through a two-step process involving spike protein binding to host receptors followed by proteolytic activation. The dendritic cell lectin \u003cem\u003eCd209\u003c/em\u003e (protein; DC-SIGN) has been reported to facilitate viral entry in certain \u003cem\u003eFIPV\u003c/em\u003e serotype II strains [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the absence of expression data from adult cats, bulk messenger RNA expression data from adult humans indicate relatively high \u003cem\u003eCd209\u003c/em\u003e expression in adipose tissue, lymph nodes, and the small intestine [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Importantly, FIPV is not primarily defined by organ-specific viral tropism but rather by its strong cellular tropism for macrophages and subsequent systemic dissemination [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consistent with this, \u003cem\u003eCd209\u003c/em\u003e is regarded as a cofactor rather than an essential receptor for FIPV entry [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Transmembrane serine protease 7 (\u003cem\u003eTmprss7\u003c/em\u003e, protein; TMPRSS7) belongs to the TMPRSS family and has been implicated in the early stages of coronavirus entry. These host proteases are reported to be important for efficient coronavirus replication [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and are involved in multiple stages of the viral life cycle, including cell entry, viral replication, protein maturation, and virion assembly [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Based on these findings, the present study investigated the expression profiles of viral receptors and \u003cem\u003eTmprss7\u003c/em\u003e in feline fetal tissues.\u003c/p\u003e \u003cp\u003eIn cats that succumbed to infection with \u003cem\u003eDabie bandavirus\u003c/em\u003e (formerly Severe fever with thrombocytopenia syndrome virus [SFTSV], family \u003cem\u003ePhenuiviridae\u003c/em\u003e), severe clinical manifestations similar to those observed in fatal human cases of SFTS have been reported. These include high fever, gastrointestinal symptoms, leukopenia, thrombocytopenia, and hepatic and renal dysfunction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, lesions in lymphoid organs have been consistently observed in both humans and cats [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similar to feline coronavirus serotype II, \u003cem\u003eCd209\u003c/em\u003e has been reported to act as a receptor involved in viral entry into dendritic cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Likewise, SFTSV shows broad organ tropism but preferentially infects cell populations such as macrophages and B-cell lineages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although transmission of SFTSV is well documented through tick bites and exposure to blood and body fluids, including human-to-human and cat-to-human transmission [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], evidence for vertical transmission remains limited in both humans and cats.\u003c/p\u003e \u003cp\u003eIn acute feline calicivirus (FCV; family \u003cem\u003eCaliciviridae\u003c/em\u003e) infection, viral antigens are most frequently detected in oral epithelial cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. FCV primarily infects the epithelial tissues of the upper respiratory tract, oral cavity, conjunctiva, and lungs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, in cats with severe systemic FCV disease, the virus can be detected in endothelial cells as well as multiple epithelial and parenchymal cell types, including oral epithelial cells, alveolar epithelial cells, epidermal cells, hepatocytes, and pancreatic acinar cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In some strains and disease presentations, FCV can cause systemic infection, with lesions involving multiple organs, including the lungs and intestines [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. During chronic infection, persistent viral shedding in oropharyngeal secretions is observed in a subset of infected cats [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, FCV has been isolated from feces, suggesting that cells in the lower intestinal tract may also support viral replication [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. FCV infection is primarily transmitted horizontally through direct contact, respiratory droplets, or contaminated environments, and there is little evidence supporting established vertical transmission from mother to offspring [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Feline junctional adhesion molecule A (\u003cem\u003eF11r\u003c/em\u003e, protein, also known as JAM-A) is a functional receptor for FCV [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In humans, \u003cem\u003eF11r\u003c/em\u003e is expressed not only in epithelial and endothelial cells but also in leukocytes and platelets and is widely distributed in barrier tissues, such as the gastrointestinal tract [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFeline morbillivirus (FeMV; family \u003cem\u003eParamyxoviridae\u003c/em\u003e) is an emerging infectious agent first identified in 2012 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similar to other morbilliviruses, it is suggested to utilize signaling lymphocyte activation molecule family member 1 (\u003cem\u003eSlamf1\u003c/em\u003e, protein; CD150) as a receptor [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eSlamf1\u003c/em\u003e has been proposed to mediate viral entry into immune cells; however, viral replication in target organs, such as the kidney, is likely to involve additional molecules beyond \u003cem\u003eSlamf1\u003c/em\u003e, and Nectin-4 has been suggested as a potential contributing factor [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In humans, \u003cem\u003eSlamf1\u003c/em\u003e is highly expressed in lymphoid tissues, including the thymus and spleen [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To date, in FeMV-positive cats, histopathological changes such as interstitial inflammatory cell infiltration, as well as degeneration and necrosis of renal tubules, have been observed in the kidney. In addition, FeMV nucleoprotein (N protein) positivity has been detected in renal tubular epithelial cells and mononuclear cells of lymph nodes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, the urinary tract has been suggested to be an important site for viral replication and persistence [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. FeMV infection is primarily transmitted via horizontal routes, with urine-mediated transmission considered one of the most likely pathways. At present, there is no clear evidence supporting vertical transmission [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The absence of detectable viremia further suggests that transplacental infection is unlikely based on current data [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, because viral RNA has also been detected in the spleen and liver, the possibility of viral replication in other organs cannot be excluded [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Indeed, in two naturally infected cats, FeMV antigen has been detected in epithelial cells of the urinary bladder, trachea, and bronchioles, as well as in lymphocytes and macrophages of the spleen and mesenteric lymph nodes and in astrocytes and oligodendroglia in the brain [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Overall, FeMV primarily targets renal tubular epithelial cells and shows tropism for lymphoid and monocytic cell lineages [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe feline panleukopenia virus (FPLV; family \u003cem\u003eParvovirinae\u003c/em\u003e) attaches to and enters host cells \u003cem\u003evia\u003c/em\u003e transferrin receptor 1 (\u003cem\u003eTfrc\u003c/em\u003e, protein; transferrin receptor 1 TfR1) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eTfrc\u003c/em\u003e is a membrane receptor involved in cellular iron uptake and is widely expressed in humans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. FPLV is highly stable in the environment and highly transmissible, and it causes severe gastrointestinal disease, particularly in young cats [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. When infection occurs during pregnancy, vertical transmission may lead to fetal infection and abortion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. FPLV preferentially targets rapidly dividing cells and replicates efficiently in tissues such as the bone marrow, lymphoid organs, and intestinal crypt epithelium [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In particular, proliferating crypt epithelial cells in the intestinal mucosa are major sites of viral replication [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, infection of lymphoid tissues results in immunosuppression through depleting susceptible cell populations [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. FPLV has also been shown to affect the developing central nervous system, including the cerebellum, during fetal development [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, domestic cat hepadnavirus (DCHBV; family \u003cem\u003eHepadnaviridae\u003c/em\u003e), first identified in 2018 as an emerging virus [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], is classified within the genus \u003cem\u003eOrthohepadnavirus\u003c/em\u003e. This genus includes the human hepatitis B virus (HBV), which is known to cause hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. DCHBV has also been suggested to be associated with hepatitis, liver cirrhosis, hepatocellular carcinoma, and other hepatobiliary disorders in cats [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Sodium taurocholate cotransporting polypeptide (\u003cem\u003eSlc10a1\u003c/em\u003e, protein; NTCP) was originally identified as a major entry receptor for HBV [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our recent study demonstrated that both human and feline \u003cem\u003eSlc10a1\u003c/em\u003e can mediate DCHBV entry [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, in humans, perinatal transmission of HBV is a major route of infection [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], with approximately 90% of infected newborns progressing to chronic infection. HBV can reach the fetus through multiple routes, including infection of placental cells and transfer via peripheral blood mononuclear cells [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Given the close genetic relationship between DCHBV and HBV, a similar mechanism of vertical transmission may also exist in cats.\u003c/p\u003e \u003cp\u003eIn this study, we quantified and compared the expression of virus receptor\u0026ndash;related genes across multiple fetal organs in cats and systematically characterized organ-specific expression patterns. Our findings demonstrate that receptor expression profiles vary substantially among organs and receptor types, with several receptors showing clear organ-specific enrichment. Notably, \u003cem\u003eSlc10a1\u003c/em\u003e exhibited a pronounced liver-specific expression pattern, whereas other receptors, such as \u003cem\u003eTfrc\u003c/em\u003e and \u003cem\u003eF11r\u003c/em\u003e, showed were broadly expressed across multiple tissues. These results provide a foundation for understanding viral organ tropism and offer insight into the potential susceptibility of fetal tissues to viral infection.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreliminary visualization\u003c/h2\u003e \u003cp\u003ePreliminary visualization revealed substantial variation in ΔCT distributions across both organs and receptors. Differences in central tendency and dispersion indicated that the data do not follow a single homogeneous distribution. In addition, measurements obtained from the same individual tended to cluster, suggesting the presence of interindividual variability. These observations supported the use of a mixed-effects modeling framework for subsequent analyses. Specifically, central tendency, variability, and the frequency of outliers differed markedly across conditions. Density plots further showed that the location and widths of distribution peaks varied by organ and receptor, reinforcing the lack of a common distributional pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Taken together, these findings provide a rationale for employing a mixed-effects model in which individual samples were treated as a random effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Histogram plots and (B) density plots showing the distribution of delta cycle threshold (ΔCT) values for each receptor gene across different organs. Columns represent organ types (brain, heart, intestine, kidney, liver, lung, spleen, and thymus), and rows represent receptor genes (transferrin receptor 1 [\u003cem\u003eTfrc\u003c/em\u003e], \u003cem\u003esignaling lymphocyte activation molecule family member 1\u003c/em\u003e [\u003cem\u003eSlamf1\u003c/em\u003e], \u003cem\u003eCd209 molecule\u003c/em\u003e [\u003cem\u003eCD209\u003c/em\u003e], \u003cem\u003ejunctional adhesion molecule A\u003c/em\u003e [\u003cem\u003eF11r\u003c/em\u003e], \u003cem\u003esolute carrier family 10 member\u003c/em\u003e [\u003cem\u003eSlc10a1\u003c/em\u003e], and \u003cem\u003eTmprss7\u003c/em\u003e). The x-axis indicates delta cycle threshold values, whereas the y-axis represents sample count in (A) and density in (B). Both visualizations demonstrate that expression distributions vary across organ types and receptor genes, with differences in modal values and dispersion. The density plots in (B) provide a smoothed representation of the data and highlight shifts in peak positions across organs and receptors.\u003c/p\u003e \u003cp\u003eDensity plots of the ΔCT values were generated to visualize expression distributions for each individual within each organ. Even within the same organ, distributions varied across samples, indicating interindividual variability. Moreover, the extent of this variability differed among organs, with certain tissues showing greater dispersion and more distinct separation of distribution peaks. These findings suggest substantial heterogeneity in ΔCT values among individuals in specific organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additional density plots overlaying ΔCT distributions for each individual by receptor demonstrated that the degree of inter-sample variability differed across receptors. In particular, \u003cem\u003eTmprss7\u003c/em\u003e exhibited a broader distribution and more pronounced shifts in peak positions across samples compared with other receptors. In contrast, the distributions for other receptors were relatively similar, indicating lower inter-sample variability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Scatter plots of ΔCT values organized by organ and receptor further showed that expression patterns differed clearly according to both factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Within the same organ, ΔCT values varied across receptors, indicating receptor-specific expression profiles. At the same time, variation in ΔCT values was observed among individuals even within the same organ\u0026ndash;receptor combinations, demonstrating interindividual heterogeneity. The magnitude of this variability differed across both organs and receptors. Notably, \u003cem\u003eTmprss7\u003c/em\u003e exhibited a broader distribution of ΔCT values compared with other receptors, suggesting greater variability among individuals. In certain organs (such as the heart), differences in the overall ΔCT levels between individuals were also evident. These patterns are more consistent with systematic interindividual differences rather than being driven by an isolated outlier. In contrast, ΔCT distributions for other receptors were more tightly clustered, indicating lower interindividual variability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Scatter plots showing delta cycle threshold (ΔCT) values across organs for each receptor gene. Panels represent receptor genes (transferrin receptor 1 [\u003cem\u003eTFRC\u003c/em\u003e], signaling lymphocyte activation molecule family member 1 [\u003cem\u003eSLAMF1\u003c/em\u003e], CD 209 molecule [\u003cem\u003eCD209\u003c/em\u003e], junctional adhesion molecule A [\u003cem\u003eF11R\u003c/em\u003e], solute carrier family 10 member 1 [\u003cem\u003eSLC10A1\u003c/em\u003e], and transmembrane serine protease 7 [\u003cem\u003eTMPRSS7\u003c/em\u003e]). The x-axis indicates organ types, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ\u0026ndash;receptor combination. (B) Scatter plots showing ΔCT values across receptor genes for each organ. Panels represent organ types. The x-axis indicates receptor genes, and the y-axis indicates ΔCT values. Each colored point represents an individual sample, and black points indicate the mean value for each organ\u0026ndash;receptor combination. These plots demonstrate that expression patterns vary across organs and receptors and reveal interindividual variability within each organ\u0026ndash;receptor combination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMixed-effects modeling\u003c/h3\u003e\n\u003cp\u003eBased on these observations, a linear mixed-effects model including sample as a random effect was fitted. Normality of the residuals was assessed using a normal Q\u0026ndash;Q plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The residuals were approximately aligned with the theoretical normal distribution, particularly in the central region, indicating an acceptable overall model fit. Although minor deviations were observed in the tails, no major departures from normality were detected. These findings suggest that the assumption of normally distributed residuals was reasonably satisfied, supporting valid statistical inference from the mixed-effects model. A residuals-versus-fitted plot was used to further evaluate model adequacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The residuals were randomly distributed around zero across the range of fitted ΔCT values, with no evident systematic patterns such as trends, curvature, or funnel-shaped dispersion. This indicates that the variance of residuals was approximately constant and that no clear violations of model assumptions, such as heteroscedasticity or model misspecification, were observed. Together with the Q\u0026ndash;Q plot, these diagnostic results support the appropriateness of the fitted mixed-effects model for subsequent statistical analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Normal Q\u0026ndash;Q plot of residuals from the linear mixed-effects model. The x-axis represents theoretical quantiles, and the y-axis represents sample quantiles of residuals (defined as observed delta cycle threshold (ΔCT) minus model-predicted ΔCT values). (B) Residuals-versus-fitted values plot from the linear mixed-effects model. The x-axis represents fitted ΔCT values, and the y-axis represents residuals. These plots were used to assess model assumptions, including normality of residuals and homoscedasticity.\u003c/p\u003e\n\u003ch3\u003ePost hoc testing\u003c/h3\u003e\n\u003cp\u003eThis analysis presents the results of post hoc comparisons based on estimated marginal means (EMMs) of ΔCT values derived from the linear mixed-effects model, with organs compared separately for each receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The EMMs were calculated after adjusting for interindividual variability by including the sample as a random effect. Each panel corresponds to a single receptor and shows differences in ΔCT values among organs within that receptor. Points represent EMMs of ΔCT, and error bars indicate the corresponding 95% confidence intervals. Letters (a\u0026ndash;d) denote statistical groupings based on Sidak-adjusted post hoc tests, with organs sharing the same letter indicating no statistically significant difference.\u003c/p\u003e \u003cp\u003eAmong the receptors examined, the most notable finding was observed for \u003cem\u003eSlc10a1\u003c/em\u003e, for which the liver exhibited ΔCT values that were significantly different from those of all other organs, indicating a pronounced organ-specific expression pattern. For \u003cem\u003eTfrc\u003c/em\u003e and \u003cem\u003eF11r\u003c/em\u003e, significant differences in ΔCT values were detected among multiple organ groups, and both receptors showed relatively high expression levels across organs overall; however, no single organ demonstrated uniquely elevated expression compared with all others. In contrast, signaling lymphocyte activation molecule family member 1 (\u003cem\u003eSlamf1)\u003c/em\u003e showed significantly higher expression in the lung, whereas \u003cem\u003eCd209\u003c/em\u003e exhibited significantly higher expression in the liver and spleen compared with other organs, highlighting receptor-specific patterns of organ enrichment. For \u003cem\u003eTmprss7\u003c/em\u003e, although statistically significant differences in ΔCT values among organs were detected, multiple organs were grouped into overlapping statistical categories. Compared with other receptors, organ-dependent differences were more moderate, and overall expression levels tended to be lower across organs.\u003c/p\u003e \u003cp\u003eTaken together, these results show that estimated marginal mean ΔCT values vary depending on both receptor and organ. The application of mixed-effects modeling framework enabled the identification of organ-specific expression patterns while appropriately accounting for interindividual variability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlots showing the estimated marginal means (EMMs) of delta cycle threshold (ΔCT) values across organs for each receptor, derived from the linear mixed-effects model. Panels represent receptor genes. The x-axis indicates organ types, and the y-axis indicates ΔCT values. Points represent EMMs adjusted for interindividual variability by including sample identity as a random effect, and error bars indicate 95% confidence intervals. Letters (a\u0026ndash;d) denote the results of post hoc comparisons among organs within each receptor, based on Sidak-adjusted EMMs. Organs sharing the same letter are not significantly different, whereas those with different letters differ significantly.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we quantified and compared the expression of viral receptor\u0026ndash;related genes across multiple fetal organs in cats. Our findings provide the first comprehensive analysis of viral receptor\u0026ndash;related gene expression across multiple organs in feline fetuses. To date, systematic comparisons of viral receptor expression across organs, particularly during the fetal stage, have rarely been limited in cats. Our analysis showed that each receptor exhibits distinct organ-specific expression patterns, offering fundamental insights into the mechanisms underlying viral organ tropism and routes of infection. These findings provide new perspectives on how viral infections are established and may help inform the assessment of tissue-specific susceptibility to infection in felines.\u003c/p\u003e \u003cp\u003eOur comprehensive analyses showed that \u003cem\u003eSlc10a1\u003c/em\u003e was highly expressed in the liver of feline fetuses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This liver-specific expression pattern is consistent with hepatotropism of DCHBV. In humans, perinatal transmission of HBV represents a major route of infection, with approximately 90% of infected newborns progressing to chronic infection. Furthermore, HBV can reach the fetus through multiple routes, including infection of placental cells and transfer via peripheral blood mononuclear cells [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Given the close genetic relationship between DCHBV and HBV, a similar mechanism of vertical transmission may also exist in cats. Notably, our study demonstrated high \u003cem\u003eSlc10a1\u003c/em\u003e expression in the fetal liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that a receptor-mediated mechanism of viral entry may be present during the fetal stage. Although there are currently no definitive reports of vertical transmission of DCHBV, these findings support the possibility of such transmission in cats. Further investigations, including analyses of \u003cem\u003eSlc10a1\u003c/em\u003e expression in adult cat placental tissues of adult cats and elucidation of maternal\u0026ndash;fetal transmission mechanisms, are warranted.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCd209\u003c/em\u003e showed enriched expression in the liver and spleen of feline fetuses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although this expression pattern is consistent with the pathogenesis of FIPV, \u003cem\u003eCd209\u003c/em\u003e is primarily expressed in specific immune cell populations, such as dendritic cells and macrophages [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].Therefore, this pattern may reflect the distribution of these cell types rather than strict organ-level specificity. Accordingly, the observed expression pattern should be interpreted in light of technical limitations, species differences, and developmental stage. Although organ-specific infection and lesion formation are influenced by additional host factors and immune responses, receptor expression in fetal tissues still provides important information regarding potential sites of viral entry. Given the reported 50%\u0026ndash;60% mortality rate of SFTS in cats [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the expression patterns observed during the fetal stage suggest that SFTSV during pregnancy may lead to fetal death. Taken together, these findings suggest that the establishment of infection and disease outcomes is governed by multiple interacting factors, among which receptor expression is an important determinant of viral susceptibility.\u003c/p\u003e \u003cp\u003eOur results showed that \u003cem\u003eTmprss7\u003c/em\u003e, a serine protease implicated in FIPV entry [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], exhibited overall low expression and no clear organ specificity in feline fetuses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that Tmprss7 is unlikely to serve as a primary determinant of infection in this context. However, a contributory role in viral entry cannot be excluded, and further functional studies are warranted.\u003c/p\u003e \u003cp\u003e \u003cem\u003eF11r\u003c/em\u003e was broadly expressed across multiple fetal cat tissues, including the heart, kidney, liver, and thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the fetus, the gastrointestinal system is still developing [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and physiological conditions differ substantially from those in postnatal individuals. Accordingly, the expression pattern observed in this study may not directly reflect that of mature animals but may instead reflect developmental-stage\u0026ndash;specific regulation. The broad expression of \u003cem\u003eF11r\u003c/em\u003e is partially consistent with the tissue distribution of FCV infection, and \u003cem\u003eF11r\u003c/em\u003e is known to function as a cell adhesion molecule in epithelial cells [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Although receptor expression alone is insufficient to determine viral tropism, efficient FCV infection may occur in epithelial environments where the receptor is exposed to the external milieu [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Therefore, given that transplacental transmission during viremia cannot be excluded, further investigation of \u003cem\u003eF11r\u003c/em\u003e protein localization is warranted.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSlamf1\u003c/em\u003e was most highly expressed in the lungs of fetal cats, with additional expression observed in the kidney and thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, the lung-dominant expression pattern observed in this study differs from that reported in humans. This discrepancy is likely attributable to the use of fetal tissues and may reflect developmental stage-dependent regulation of gene expression. Fetal organisms are exposed to limited external antigens and exist in a physiological environment distinct from that of postnatal individuals [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, SLAMF1 expression in fetal tissues may reflect developmental stage\u0026ndash;specific immune functions rather than mature activity, and the high expression observed in the lung may represent a preparatory mechanism for postnatal exposure to environmental antigens. Furthermore, FeMV infection is not thought to rely on a single receptor but instead involves a complex entry process mediated by multiple molecules, including Nectin-4 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Accordingly, \u003cem\u003eSlamf1\u003c/em\u003e expression in fetal tissues is likely associated with developmental processes of the immune system.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTfrc\u003c/em\u003e was broadly expressed across multiple fetal feline tissues, with relatively high expression in the spleen and liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The expression pattern observed in this study is generally consistent with that reported in humans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], suggesting that the basic expression profile of \u003cem\u003eTfrc\u003c/em\u003e is conserved across species. The relatively high expression of \u003cem\u003eTfrc\u003c/em\u003e, particularly in the spleen, a key hematopoietic organ during fetal development, is broadly consistent with the known tissue tropism of FPLV. In contrast, \u003cem\u003eTfrc\u003c/em\u003e expression in the fetal brain, which is also susceptible to FPLV infection, was relatively low. This may reflect tissue-specific regulatory mechanisms associated with the unique physiological environment and developmental status of the fetal brain rather than serving as a direct determinant of viral tissue tropism.\u003c/p\u003e \u003cp\u003eWhile we comprehensively analyzed the expression of multiple viral receptor genes in feline fetuses, this study has several limitations. First, because RNA was extracted from whole fetal organs, it was difficult to completely exclude the contribution of circulating blood cells. As a result, some of the observed immune-related gene expression may have been influenced by hematopoietic cells present in the vasculature. Second, the exact gestational age (weeks of pregnancy) of each fetus was not determined, preventing rigorous comparison of receptor expression across defined developmental stages and limiting interpretations of stage-dependent variation. Third, due to the small size of fetal organs, it was not possible to separate epithelial, stromal, and endothelial compartments during tissue sampling. Consequently, the expression data represent average signals across whole organs, and the assessment of microanatomical localization was not feasible. Nevertheless, this study provides the first comprehensive analysis of multiple viral receptor genes in feline fetuses, thereby contributing to a better understanding of the potential mechanisms underlying vertical transmission of viral infections.\u003c/p\u003e \u003cp\u003eRegarding future directions, it will be necessary to precisely determine fetal age and conduct longitudinal analyses to clarify developmental stage-dependent changes in receptor expression. It will also be important to investigate whether single-nucleotide polymorphisms in receptor genes are associated with viral susceptibility or organ specificity. Since FPLV is known to cause transplacental infection, assessing receptor expression in placental and maternal tissues may help elucidate the molecular mechanisms underlying vertical transmission. Moreover, this study was limited to RNA-level analysis, and whether the corresponding proteins are actually expressed remains unknown\u0026mdash;representing an important limitation. Even when receptor messenger RNA is detected, posttranslational regulation or translational suppression may prevent protein expression [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Therefore, future studies should include verification at the protein level, such as immunohistochemistry or proteomic approaches, to confirm receptor expression and tissue distribution.\u003c/p\u003e \u003cp\u003eIn conclusion, this study quantitatively characterized the expression of viral receptor\u0026ndash;related genes across multiple fetal organs in cats and demonstrated that receptor expression profiles vary markedly across both organs and receptor types. Distinct organ-specific enrichment was observed for several receptors, including liver-specific expression of \u003cem\u003eSlc10a1\u003c/em\u003e, whereas other receptors, such as \u003cem\u003eTfrc\u003c/em\u003e and \u003cem\u003eF11r\u003c/em\u003e, showed broader tissue distribution. These findings support our initial hypothesis that receptor expression is organ-dependent and provide a framework for understanding viral organ tropism. While receptor expression alone is insufficient to determine infection outcomes, our results suggest that it is an important factor contributing to viral susceptibility, particularly in the context of DCHBV. Future studies incorporating developmental stage, cellular resolution, and protein-level validation will be essential to further elucidate the mechanisms underlying viral infection and pathogenesis in felines.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving the collection of\u0026nbsp;feline\u0026nbsp;samples were approved by the Animal Care and Use Committee of the University of Miyazaki (Approval No.\u0026nbsp;2023-012) and were conducted in accordance with the University of Miyazaki experimentation regulations and relevant institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Research: Reporting of In Vivo Experiments statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is reported in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines. No live animals were used specifically for this study. The fetal tissues analyzed were obtained from fetuses that were already dead at the time of collection from a surgically removed uterus. Therefore, no euthanasia or experimental animal procedure was performed for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrgan collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrgan collection was performed as follows. The uterus was obtained from a cat that underwent ovariohysterectomy for medical reasons at a university-affiliated veterinary hospital in Japan and was kept on ice until use. The fetuses were already dead at the time the uterus was collected. Therefore, no animals were euthanized for the purposes of this study, and no euthanasia method or anesthetic agent for fetal sacrifice was applicable.\u0026nbsp;The uterus was dissected to retrieve the fetuses. From each fetus, tissue samples of the lung, intestine, spleen, kidney, heart, brain, liver, and thymus were carefully excised using sterile scissors and forceps. Each tissue sample was immediately placed into a 1.5 mL microcentrifuge tube containing RNAlater Stabilization Solution (Thermo Fisher Scientific, catalog no. AM7020) and stored at −30°C until RNA extraction. No predetermined gestational age was available for the fetuses, and the age of the mother cats was also unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were initially disrupted using a Biomasher\u0026nbsp;(Nippi, Inc., catalog no. 893064).\u0026nbsp;The homogenized tissues were then lysed in Buffer RLT supplemented with 2-mercaptoethanol (Bio-Rad, Hercules, CA, USA, Catalog no. 1610710). RNA was subsequently purified according to the RNeasy Mini Kit protocol\u0026nbsp;(Qiagen, catalog no. 74104). RNA was eluted with 90 μL of RNase-free water, and RNA concentrations were measured using a NanoDrop Eight spectrophotometer (Thermo Fisher Scientific, Inc., catalog no. NDE-GL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative real-time polymerase chain reaction was performed using the One Step TB Green® PrimeScript™ PLUS Real-Time Polymerase Chain Reaction Kit (Takara Bio Inc., catalog no. RR096A) on a QuantStudio5 Real-Time Polymerase Chain Reaction System (Thermo Fisher Scientific). Primers listed in\u0026nbsp;\u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e were used in this study. Gene expression levels were quantified using the comparative ΔCt method. ΔCt values were calculated as Ct(target gene) – Ct (\u003cem\u003eribosomal protein S7\u003c/em\u003e (\u003cem\u003eRps7\u003c/em\u003e)), where \u003cem\u003eRps7\u003c/em\u003e was used as the reference housekeeping gene [59]. All qRT-PCR reactions were performed in technical triplicate. For each sample, triplicate Ct values were inspected for consistency. When all three replicates produced valid Ct values, the mean Ct was used for subsequent analysis. If one replicate showed clear deviation from the others (e.g., due to technical error), it was excluded, and the mean of the remaining replicates was used. Samples with insufficient valid replicates were treated as missing data. Undetermined Ct values were treated as missing values and were not imputed. These missing observations were retained as NA in downstream analyses. Potential outliers were initially identified based on technical inconsistency and statistical criteria (see below). However, outliers were not removed from the primary analysis unless there was clear evidence of technical error. Sensitivity analyses were performed with selected outliers removed to assess the robustness of the results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo account for the hierarchical structure of the data, a linear mixed-effects model was fitted using the lme4 package in R. To account for the hierarchical structure of the data, a linear mixed-effects model was fitted using the lme4 package in R. Organ and receptor were treated as fixed effects, and their interaction term (organ × receptor) was included in the model. Sample identity (individual cat) was included as a random intercept to account for repeated measurements obtained from the same individual. Model comparison was performed to assess the contribution of the interaction term by comparing models with and without the interaction using likelihood ratio tests. Post hoc comparisons among organs within each receptor were conducted using estimated marginal means implemented in the emmeans package. Multiple comparisons were adjusted using Sidák correction within each receptor. Model assumptions were evaluated using residual-versus-fitted plots to assess homoscedasticity and Q–Q plots to assess normality of residuals. A p-value of 0.05 or less was considered statistically significant. All analyses were conducted using R version 4.5.2 (2025-10-31).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Ms. Tomoko Nishiuchi, Ms. Miki Kawano, Ms. Natsumi Matsubara, and the staff of CADIC, University of Miyazaki, for their assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAH.K., and A.S. designed the experiments. AH.K., and NX.K performed the experiments. AH.K., NX.K, Z.M, A.O-A, and A.S. analyzed the results. M.S., Z.M., A.O-A., and A.S. wrote and edited the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource data are available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Japan Agency for Medical Research and Development (AMED) Research Program on HIV/AIDS JP26fk0410075, JP26fk0410080, JP25fk0410056, and JP25fk0410058 (to A.S.); the AMED Program for Accelerating Medical Research JP256f0137007j0001 (to A.S.); the JSPS KAKENHI Grant-in-Aid for Scientific Research (C) JP24K09227 (to A.S.); the JSPS KAKENHI Grant-in-Aid for Scientific Research (B) JP22H02500 (to A.S.) and JP21H02361 (to A.S.); the JSPS Bilateral Program JPJSBP120245706 (to A.S.); the JSPS Fund for the Promotion of Joint International Research (International Leading Research) JP23K20041 (to A.S.); and the G-7 Grants (2025 and 2026) (to A.S.). This study was supported by the Frontier Science Research Center, University of Miyazaki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving the collection of\u0026nbsp;feline\u0026nbsp;samples were approved by the Animal Care and Use Committee of the University of Miyazaki (approval No. 2023-012) and conducted in accordance with the University of Miyazaki Experimentation regulations and relevant institutional guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAsami Oguro-Ando and Akatsuki Saito\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTasker, S. \u003cem\u003eet al.\u003c/em\u003e Feline Infectious Peritonitis: European Advisory Board on Cat Diseases Guidelines. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eVennema, H., Poland, A., Foley, J. \u0026amp; Pedersen, N. 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C. \u003cem\u003eet al.\u003c/em\u003e A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues. \u003cem\u003eVet Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e120\u003c/strong\u003e, 212\u0026ndash;222 (2007).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"viral receptor, organ tropism, gene expression, linear mixed-effects model, tissue-specific expression","lastPublishedDoi":"10.21203/rs.3.rs-9442451/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9442451/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral receptors play a key role in determining viral organ tropism and host susceptibility to infection. However, distribution of viral receptor expression across feline organs, particularly during the fetal stage, remains poorly understood. In this study, we examined the expression patterns of multiple viral receptor\u0026ndash;related genes across fetal cat organs using quantitative polymerase chain reaction. Gene expression levels were quantified as delta cycle threshold values and analyzed using a linear mixed-effects model to account for repeated measurements from the same individual. Initial data visualization showed considerable variation in delta cycle threshold values across different organs and receptors, along with interindividual variability. Statistical analysis using mixed-effects modeling confirmed significant differences in expression patterns among organs and receptors. Notably, several receptors exhibited pronounced organ-specific expression patterns, whereas others showed broader distributions across tissues. In addition, variability among individuals was observed across organ\u0026ndash;receptor combinations, highlighting heterogeneity in receptor expression among fetal samples. These findings indicate that viral receptor expression in feline fetal tissues is both organ-specific and subject to interindividual variation. Overall, these results provide a foundational dataset for understanding routes of viral entry and organ tropism in cats and may support future research into feline viral pathogenesis and vertical transmission.\u003c/p\u003e","manuscriptTitle":"Multi-Organ Expression of Viral Entry Receptors in Feline Fetal Tissues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 15:47:07","doi":"10.21203/rs.3.rs-9442451/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"311582559641444981667586621562783818999","date":"2026-05-09T21:00:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335569589992053392356327573268145154894","date":"2026-05-05T23:28:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2080034783935119075017401563563117351","date":"2026-04-30T09:44:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-26T01:45:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-25T21:27:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-24T10:27:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-21T08:39:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-21T08:27:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff6abea6-c5cb-456c-9a1a-93091ff9deda","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"311582559641444981667586621562783818999","date":"2026-05-09T21:00:26+00:00","index":67,"fulltext":""},{"type":"reviewerAgreed","content":"335569589992053392356327573268145154894","date":"2026-05-05T23:28:21+00:00","index":64,"fulltext":""},{"type":"reviewerAgreed","content":"2080034783935119075017401563563117351","date":"2026-04-30T09:44:16+00:00","index":56,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67854339,"name":"Biological sciences/Biological techniques"},{"id":67854340,"name":"Biological sciences/Computational biology and bioinformatics"}],"tags":[],"updatedAt":"2026-05-12T15:47:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 15:47:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9442451","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9442451","identity":"rs-9442451","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}