Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice

Yuanjiu Miao; Yue Zheng; Ting Wang; Wenfu Yi; Nailou Zhang; Wanpo Zhang; Zhenhua Zheng

doi:10.1128/jvi.01709-23

. 2024 Feb 2;98(3):e01709-23. doi: 10.1128/jvi.01709-23

Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice

Yuanjiu Miao ^1,^2,^#, Yue Zheng ^1,^2,^#, Ting Wang ^1,³, Wenfu Yi ^1,², Nailou Zhang ¹, Wanpo Zhang ^4,^✉, Zhenhua Zheng ^1,^✉

Editor: Mark T Heise⁵

PMCID: PMC10949448 PMID: 38305156

ABSTRACT

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup’s stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.

IMPORTANCE

To date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

KEYWORDS: tick-borne flavivirus, breast milk, infection routes, mammary glands, bioluminescence imaging

INTRODUCTION

Tick-borne encephalitis virus (TBEV) is the most important human pathogen among tick-borne flaviviruses (TBFs) and is responsible for causing tick-borne encephalitis (TBE), a virulent neurological zoonotic disease in humans (1). TBE cases are predominantly reported in eastern, central, and northern Europe; northern China; Mongolia; and Russia (2, 3), and specifically, the highest incidence of TBE (>10/100,000 population) is found in Baltic nations, the Czech Republic, Russia, and Slovenia (1, 4). However, despite effective vaccination options, over 3,000 cases were reported in Europe in 2019, with a 0.7% case fatality rate (5). TBEV is primarily transmitted via tick bites; however, its milk-borne transmission has become a growing concern (6 –8). Historically, the first milk-borne outbreak of TBE occurred in the Roznava District of Slovakia between 1951 and 1952, resulting in at least 660 cases. Since then, milk-borne epidemics and isolated cases have been documented in Eastern Europe, Austria, and Germany. Moreover, between 2007 and 2011, 29 typical TBE cases and 4 TBE cases due to ingested contaminated foods were diagnosed in Hungary (9, 10); likewise, six individuals were infected with TBEV after consuming infected goat cheese in Austria (11). Furthermore, between 2007 and 2016, 26 alimentary outbreaks of TBE were reported, with raw milk consumption contributing to 17% of all TBEV infections during that period (12). Thus, these outbreaks underscore the importance of considering TBEV transmission via foodborne routes.

Although the exact mechanism of TBEV transmission through the alimentary route is not yet clearly understood, the virulence of the virus may be influenced by the digestion of dairy products in the gastrointestinal tract. In particular, studies have shown that experimentally infected goats excrete TBEV up to 8 days post-infection (dpi) and exhibit TBEV infection with the virus detectable in their small intestines following oral infection (10, 13, 14). Usually, after ingestion, milk reaches the duodenum within a few minutes and after 1.5–2 h, it empties from the stomach (15). Consistent with this occurrence, Pogodin et al. reported that TBEVs maintain their infectivity in normal gastric juice (pH: 1.49–1.80) for up to 2 h (16). However, while researchers postulated that TBEV probably enters the organism via small intestinal M cells of the Peyer’s patches, which transport viral particles to the intestinal lymphoid tissue (17), experimental evidence is lacking. In addition, in a study on the Kyasanur Forest disease virus, which is also a part of the TBEV group, the virus antigen was detected in the epithelial cells of the gut mucosa in bonnet macaques (18). Thus, these findings implied that TBEVs were likely released into the circulatory system and subsequently transferred to various organs, such as the brain (19).

Mother-to-child transmission during breastfeeding has been demonstrated for the Zika virus (ZIKV), another flavivirus that can cross the intestinal barrier in experimental models (20). Although alimentary infections through raw milk and dairy products from infected animals have been known since the 1950s, current knowledge on the mechanism of TBEV infection during breastfeeding and the dynamics of mother-to-child transmission is limited and often based on early experimental studies. However, in 2020, a case of mother-to-child TBEV infection was reported, indicating the possibility of interhuman transmission of TBEV through breastfeeding (21). Despite the potential threat of milk-borne transmission of TBFs, laboratory models that investigate the detailed processes involved are lacking.

Therefore, to confirm milk-borne transmission of TBFs in this study, we used the naturally attenuated tick-borne flavivirus Langat virus (LGTV) strain TP21 as a model because LGTV shares 82–88% amino acid identity with TBEV but does not cause human disease (22, 23). In addition, as bioluminescence enabled by luciferase-based reporters with high signal-to-background ratios is commonly used for noninvasive imaging of deep tissues in live animal studies (24), we generated an LGTV infectious clone of strain TP21 and a bioluminescent NanoLuc reporter virus (Nluc-LGTV) to study the different transmission routes of TBFs in mice. Using this reporter virus, we investigated the alimentary infection route and compared the different routes of LGTV infection in mice. Furthermore, we also investigated infectious LGTV in mouse milk and mammary glands as well as that in mammary gland epithelial cells. We hypothesized that our findings would suggest the possibility of mother-to-child transmission of tick-borne flaviviruses and provide insights into the transmission process.

RESULTS

Generation of Nluc-LGTV reporter virus

To investigate the transmission routes of the tick-borne flavivirus, we generated an infectious clone and a Nluc-LGTV reporter virus (Fig. 1A). We cloned the full-length genome of the LGTV TP21 strain into the vector pACYC177, inserting an intron in the viral NS1 gene to improve plasmid stability in Escherichia coli (25). To construct the Nluc-LGTV, we inserted the Nluc gene after the N-terminal 68 amino acids (AA) of the C gene (C68), with a porcine teschovirus-1 2A protease gene (P2A) inserted between Nluc and the full-length C gene to ensure the release of functional Nluc and C proteins. To prevent homologous recombination between the full-length C gene and C68, we introduced ORF-shifting mutations with a single A nucleotide insertion at C8 and G nucleotide deletion at C68 (26).

We recovered both the wild-type (WT) LGTV and the Nluc-LGTV reporter viruses in BHK-21 cells by transfecting infectious clone plasmids. However, we found that the Nluc gene was unstable in the LGTV genome and was completely lost after six passages (Fig. 1B). To obtain a stable Nluc-LGTV reporter virus, seven rounds of plaque purification were performed (Fig. 1C). The purified Nluc-LGTV had a smaller plaque size than the wild type (Fig. 1D and E). After 10 passages in BHK-21 cells, we confirmed the stable anchoring of the Nluc gene in the LGTV genome by reverse transcription-PCR (RT-PCR) (Fig. 1F). An immunofluorescence assay using a TBEV E protein antibody showed that both WT-LGTV and Nluc-LGTV could be detected in infected BHK-21 cells (Fig. 1G). The viral titers of Nluc-LGTV were lower than those of WT-LGTV at 12, 24, and 48 h post-infection, suggesting that the reporter virus had a lower replication capacity in BHK-21 cells than that of the WT virus (Fig. 1H). However, we detected a high expression level of Nluc luciferase from Nluc-LGTV-infected BHK-21 cell lysis through luciferase activity assay, with the peak of Nluc expression occurring at 72 h post-infection (Fig. 1I).

In vivo characterization of Nluc-LGTV

To assess the in vivo pathogenicity of WT-LGTV versus Nluc-LGTV, we used the A129 mouse strain, which lacks type I interferon (IFN-α/β) receptors and serves as an appropriate animal model of LGTV infection (22). After intraperitoneal infection with a range of viral doses, we monitored the body weight and survival rate of the mice for 21 dpi. Mice infected with WT-LGTV succumbed to viral infection at doses as low as 10 PFU (Fig. 2A). In contrast, Nluc-LGTV demonstrated lower pathogenicity in A129 mice, with a 50% lethal dose (LD₅₀) of 10^2.503 PFU (Fig. 2A). Both WT-LGTV and Nluc-LGTV infections led to viremia in mice, but lower viral RNA loads were detected in the serum of Nluc-LGTV-infected mice (Fig. 2B). The organs of WT-LGTV or Nluc-LGTV infected mice were harvested on the corresponding days of peak viral RNA (D4 for WT-LGTV and D5 for Nluc-LGTV). Similarly, the viral loads of Nluc-LGTV were lower than those of WT-LGTV in every organ examined (Fig. 2C). We used ex vivo imaging to visualize the distribution of Nluc-LGTV in various organs isolated from infected mice. Significant Nluc signals were detected in all organs compared to the uninfected group. As shown in Fig. 2D and E, high Nluc signals were observed in the heart, liver, spleen, lung, kidney, gastrointestinal tract, testis, and uterus of infected mice. However, Nluc signals were weakly detected in the brain, possibly because of poor substrate permeability across the blood-brain barrier (27).

Fig 2 — *In vivo* characterization of Nluc-LGTV. (A) Survival analysis of 6–7 weeks old A129 mice following intraperitoneal injection with PBS, 10³, 10², and 10 PFU of WT-LGTV (WT) or Nluc-LGTV (Nluc) (N = 6). (**B, C**) A129 mice [6–7 weeks old; N = 4 (two females, two males)] were intraperitoneally injected with 10⁵ PFU of WT-LGTV (WT) or Nluc-LGTV (Nluc). (B) Serum viral loads were quantified at the indicated days post-infection (dpi) by RT-qPCR. The dashed line indicates the limit of detection (10³ copies/mL) (B). Organ viral loads were quantified at 4 (WT) or 5 (Nluc) dpi by RT-qPCR. The dashed line indicates the limit of detection (50 copies/mg) (C). Data represent the standard error of the mean (SEM) analyzed by two-way ANOVA (ns, not significant; ***, P < 0.001; ****, P < 0.0001). (**D, E**) A129 mice (6–7 weeks old) were intraperitoneally injected with PBS [Uninfected; N = 4 (two females, two males)] or 10⁵ PFU of Nluc-LGTV [Infected; N = 5 (two females, three males)]. Organs including heart (H), liver (L), spleen (S), lung (Lu), kidney (K), uterus (U), testis (T), brain (B), and gastrointestinal tract were subjected to *ex vivo* bioluminescence imaging (D). The average radiance was determined by ROI analysis. Data represent the mean ± SEM analyzed by two-way ANOVA (**, P < 0.01; ****, P < 0.0001) (E).

LGTV infection in mice via the intragastric route

To investigate whether LGTV can infect mice via the gastrointestinal tract and to evaluate the characteristics of different infection routes, we inoculated mice with the same dose of WT-LGTV (10⁵ PFU) via intragastric gavage (IG), intraperitoneal injection (IP), or footpad injection (FP) and monitored their body weight and survival rate. We found that compared to those in the IP and FP routes, weight loss and mortality occurred at a later time via the IG route (Fig. 3A and B).

Fig 3 — Characterization of LGTV infection routes. (**A, B**) A129 mice (6–7 weeks old; N = 6) were inoculated with 10⁵ PFU LGTV via the intragastric gavage, intraperitoneal injection, or footpad injection. Mice were monitored for 8 days for body weight loss (A) and survival (B). (**C–F**) A129 mice (6–7 weeks old) were inoculated with PBS (Uninfected; N = 2) or 10⁵ PFU Nluc-LGTV via the footpad injection (N = 5) or intragastric gavage (N = 4) routes. Ventral and dorsal views of Nluc-LGTV-infected mice were monitored in real time at the indicated intervals (C). The average radiance was determined by region of interest (ROI) analysis of the ventral or dorsal sides. Data represent the mean ± SEM (**D, E**). Mice were monitored for survival (F).

To assess the infection efficiency of Nluc-LGTV via different routes, we infected mice with 10⁵ PFU Nluc-LGTV via FP or IG routes and measured luminescence intensities of mice’s ventral and dorsal sides using an in vivo imaging system (IVIS) after infection (Fig. 3C). As shown in Fig. 3C, in mice infected by the FP route, distinct Nluc expression could be detected as early as 2 dpi, compared to that in uninfected mice, and the luminescence intensity increased dramatically within a few days of infection. Conversely, in mice infected via the IG route, significant Nluc intensity was observed at 5 dpi, and the Nluc intensity rapidly accumulated over time. During the quantitative analyses of IG infection, luminescence intensity showed a lower and delayed curve compared to that in the FP route, both on the ventral and dorsal sides (Fig. 3D and E). The mortality occurred at 6 and 7 dpi in the FP route group, and at 9 and 10 dpi in the IG route group (Fig. 3F).

Infectious LGTV present in mice milk and mammary glands

To investigate the presence of infectious LGTV in milk and mammary glands, we infected dams with LGTV (1 × 10⁵ PFU) 2 days after parturition. The milk from LGTV-infected dams was collected at 1 and 2 dpi. Infectious LGTV (4 × 10⁵ to 2 × 10⁸ PFU/mL) was detected in milk at 2 dpi (Fig. 4D). The dams were too weak to collect milk at 3 dpi, so we isolated mammary glands to detect the LGTV titer of tissue suspension. As is shown in Fig. 4A, 1 × 10⁷ to 4 × 10⁷ PFU/g LGTV were detected in tissue suspension of mammary glands. In addition, strong expression of Nluc was observed in the mammary glands of Nluc-LGTV-infected mice (Fig. 4B and C). To further assess the infectivity of LGTV in milk, we collected milk from LGTV-infected dams at 2 dpi, which were incubated with BHK-21 cells at an MOI of 1 for 72 h. An immunofluorescence assay (IFA) using a TBEV E protein antibody detected the presence of LGTV in BHK-21 cells (Fig. 4E).

Fig 4 — Presence of LGTV in mice mammary glands and milk. (A) A129 dams (15–20 weeks old; N = 6) were intraperitoneally injected with 10⁵ PFU of LGTV. The viral loads of mammary glands were determined at 3 dpi by plaque assay. Data represent the mean ± SEM. The dashed line indicates the limit of detection (250 PFU/mL). (**B, C**) Female A129 mice (6–7 weeks old; N = 3) were intraperitoneally injected with PBS (Uninfected) or 10⁵ PFU of Nluc-LGTV (Infected). Mammary glands were subjected to *ex vivo* bioluminescence imaging (B). The average radiance was determined by ROI analysis of the mammary glands. Data represent the mean ± SEM analyzed using an unpaired t test (***, P < 0.001; N = 3) (C). (**D, E**) A129 dams (15–20 weeks old; N = 6) were intraperitoneally injected with PBS (Uninfected) or 10⁵ PFU of LGTV (Infected). The viral loads of mice milk were determined at 1 or 2 dpi by plaque assay. Data represent the mean ± SEM analyzed by an unpaired t test (****, P < 0.0001; N = 6). The dashed line indicates the limit of detection (666.7 PFU/mL) (D). BHK-21 cells were incubated with milk from uninfected mice or LGTV-infected mice at an MOI of 1 for 72 h. IFA analysis of E protein expression in BHK-21 cells. Cells were fixed and immunostained with TBEV E protein hyperimmune rabbit serum (red). Cell nuclei were stained with Hoechst 33258 (blue) (scale bars: 25 µm) (E). (**F, G**) A129 mice (6–7 weeks old; N = 6) were inoculated with milk from uninfected mice or LGTV-infected mice (4 × 10⁵ PFU) via the intragastric gavage. Mice were monitored for 14 days for body weight loss (F) and survival (G).

We also administered the milk of LGTV-infected mice (4 × 10⁴ PFU) intragastrically to adult mice and monitored their body weight and survival rate. As shown in Fig. 4F and G, weight loss began at 4 dpi and the weight recovered between 6 and 8 dpi, because mortality occurred between 6 and 13 dpi. The inconsistent progression of infection in different mice may influence the average body weight when mice numbers change. These data confirm the infectiousness of milk from LGTV-infected dams, and provide evidence of the potential transmission of LGTV through the consumption of contaminated milk.

Dam-to-pup infection of LGTV through breastfeeding

To investigate the potential dam-to-pup transmission of LGTV through breastfeeding, we infected dams with LGTV (1 × 10⁵ PFU) 2 days after parturition and monitored their survival rate (Fig. 5B). Mortality occurred between 4 and 5 days post-infection. We detected LGTV viral RNA in SMCs and the blood of pups using quantitative reverse transcription PCR (RT-qPCR) (Fig. 5C and D), indicating transmission occurred via suckling.

Fig 5 — Dam-to-pup transmission of LGTV in mice. (A–D) The A129 dams (15–20 weeks old; N = 6) were intraperitoneally injected with PBS (Uninfected) or 10⁵ PFU of LGTV (Infected). Schematic representation of the detection of dam-to-pup transmission of LGTV in mice (A). Dams were monitored for 5 days for survival (B). The viral loads of stomach milk clots (SMCs) of pups were determined in the uninfected group (N = 8) and infected group at 1 (N = 8), 2 (N = 9), and 3 (N = 5) dpi using RT-qPCR. The dashed line indicates the limit of detection (100 copies/ug RNA). Data represent the standard error of the mean (SEM) analyzed by two-way ANOVA (ns, not significant; ***, P < 0.001; ****, P < 0.0001) (C). The viral loads in pups' blood were determined in the uninfected group (N = 6) and infected group at the indicated dpi (N = 6) using RT-qPCR. The dashed line indicates the limit of detection (1000 copies/µg RNA). Data represent the standard error of the mean (SEM) analyzed by two-way ANOVA (ns, not significant; ****, P < 0.0001) (D). (E–H) Three groups of A129 mice were monitored for the dynamics of infection by *in vivo* imaging. Dams (15–20 weeks old; N = 3) were infected intraperitoneally with 10⁵ PFU of Nluc-LGTV. Schematic representation of the bioluminescence imaging of dam-to-pup transmission of LGTV in mice (E). Viral spreads of Nluc-LGTV-infected dams and pups were monitored in real-time at the indicated times (F). The average radiance was determined by the ROI analysis of dams (G). Pups were monitored for 12 days for survival (group 1, N = 7; group 2, N = 8; and group 3, N = 8) (H).

To further investigate dam-to-pup transmission through breastfeeding, we infected three dams with 10⁵ PFU Nluc-LGTV 2 days after parturition and monitored viral loads by IVIS every 2 days post-infection. All dams survived the viral infection, and luminescence intensity peaked at 4 dpi (groups 1 and 3) or 6 dpi (group 2) before significantly decreasing (Fig. 5F and G). At 10 dpi, luciferase signal was detected in pups by IVIS, with six pups infected in group 1, two in group 2, and none in group 3 (Fig. 5F). The survival rate of the infected pups was 0% for group 1, 62.5% for group 2, and 100% for group 3 (Fig. 5H). These findings confirm that LGTV can be transmitted from infected dams to their offspring during breastfeeding, resulting in high mortality rates in pups.

LGTV infects epithelial cells of the mammary gland

Given that LGTV was detected in the mammary gland, we investigated the involvement of this gland in LGTV infection. Histopathological analysis of hematoxylin and eosin (H&E)-stained mammary glands from LGTV-infected mice revealed epithelial cell disorder, luminal cell necrosis and shedding, connective tissue hyperplasia, and eosinophilic infiltration (Fig. 6A through D). Immunohistochemistry (IHC) staining using an LGTV E protein antibody, the LGTV E protein was detected near the mammary gland epithelial cells (Fig. 6E through H). Additionally, LGTV viral particles were observed around the endoplasmic reticulum (ER) membranes and in an intracellular vesicle of LGTV-infected mice mammary glands by electron microscopy (Fig. 6I and J). Furthermore, to determine which types of epithelial cells in the mammary gland were targeted by the LGTV, we used Krt8 and Krt14 as markers for luminal and myoepithelial cells, respectively (28). We labeled the cells with antibodies for these markers and used the LGTV E protein antibody as a viral marker. Immunofluorescence observations revealed co-localization of the viral E protein with both Krt8 and Krt14, indicating LGTV infection occurred near or in the luminal and myoepithelial cells (Fig. 6K). Thus, these results suggest that LGTV can infect within different types of mammary gland epithelial cells. The observed histopathological changes and cell tropism provide insights into the pathogenesis of LGTV in mammalian hosts.

Fig 6 — Identification of LGTV-infected cells in mice mammary gland. The A129 dams were injected intraperitoneally with PBS (Uninfected) or 10⁵ PFU LGTV (Infected). (A–D) H&E staining of mammary glands from uninfected (A) and LGTV-infected mice (**B–D**) at 5 dpi (scale bars: 200 µm or 50 µm). Luminal cell necrosis and shedding (black arrow), eosinophilic infiltration (blue arrows) (C). Epithelial cell disorder (black arrow) and connective tissue hyperplasia (blue arrow) (D). (E–H) Expression of the E protein in mammary glands from uninfected (E) or LGTV-infected (**F–H**) mice at 5 dpi. The mammary glands were stained with rabbit anti-LGTV EDIII by immunohistochemistry (scale bars: 200 µm or 50 µm). (I and J) Transmission electron micrographs of mammary glands from LGTV-infected mice at 5 dpi. LGTV viral particles around ER membranes (scale bars: 5 µm, 1 µm, or 100 nm) (I). LGTV viral particles in an intracellular vesicle (scale bars: 2 µm or 200 nm) (J). (K) Immunofluorescence staining of mammary glands from uninfected and LGTV-infected mice with anti-LGTV EDIII (green), Krt8 (red), and Krt14 (red). Cell nuclei were stained with DAPI (blue) (scale bar: 20 µm). (L) Schematic of luminal structure.

DISCUSSION

TBEV has been reported in several instances of human infections associated with the consumption of raw milk, cheese, or dairy products (17). Studies have detected TBEV in unpasteurized goat cheese (6) and shown that LGTV can remain stable in goat milk for several days at 4°C; however, it declines significantly after 24 h at room temperature and is undetectable after 48 h (29). Recently, a probable case of TBEV transmission from an unvaccinated mother to her infant through breastfeeding was reported (21). However, confirming breast milk transmission of TBEV has been challenging because of the lack of a suitable animal model. Therefore, to investigate the possibility of breast milk transmission in TBF, type I interferon (IFN-α/β) receptor-deficient A129 mice were used as a model of TBF infection in this study. Furthermore, similar to breast milk transmission studies of ZIKV (30, 31), milk and breast tissue samples were collected from LGTV-infected dams, and high titers of LGTV in both samples were detected by plaque assays. Moreover, LGTV viral RNA was detected in the serum and SMC of pups, thus, indicating the potential for TBF transmission via breastfeeding.

Currently, a mechanistic understanding of the milk-borne transmission of TBF is lacking. In this study, we found that milk from LGTV-infected mice could independently infect adult mice via intragastric gavage. Furthermore, evidence of the involvement of mammary glands in TBF transmission is limited. We observed significant breast tissue lesions, epithelial cell disorder, luminal cell necrosis and shedding, connective tissue hyperplasia, and eosinophilic infiltration in the mammary glands of the infected mice (Fig. 6A through D). Immunohistochemistry and immunofluorescence assays showed the presence of LGTV E protein near or in both luminal and myoepithelial cells (Fig. 6E through H and K), providing further evidence for the transmission of TBF through the mammary gland. Besides, transmission electron microscopy (TEM) images supported the presence of viral particles in the mammary glands of the LGTV-infected mice (Fig. 6I and J). Consistent with our findings regarding the presence and dynamic changes of the LGTV in the mammary gland, previous studies have reported the presence of high levels of infectious ZIKV as early as 3 and 6 dpi in the mammary glands of infected mice, indicating rapid dissemination of the virus from the bloodstream to the mammary glands and productive infection occurring in mouse mammary glands (30, 32). Furthermore, we have demonstrated, using bioluminescent imaging in vivo, the significance of the mammary gland in viral infection and transmission of the virus through breast milk (Fig. 4).

Compared with other TBF infection routes, such as FP and IP infections, gastrointestinal tract infection with LGTV resulted in a longer duration of infection and a lower rate of weight loss (Fig. 3A). This finding may be attributed to the low pH environment of the gastrointestinal tract, which could potentially decrease viral infectivity. Nonetheless, our findings demonstrated that LGTV could still establish an infection in the gastrointestinal tract, as evidenced by the high viral loads in the gastrointestinal tract (Fig. 2B through E). Moreover, studies on TBEV have also shown that it can remain infectious in gastric juice (pH: 1.49–1.80) for up to 2 h when mixed with milk, indicating a potential route of transmission through the gastrointestinal tract (33, 34). In particular, we observed that the virus is transmitted through the intestinal M cells of Peyer’s patches, which transport the virus into the intestinal lymphoid tissue where primary replication occurs. However, a recent study reported that LGTV could not infect immunodeficient mice via oral gavage, with or without milk (35). Therefore, we speculate that this discrepancy may be due to differences in the experimental setup, such as the LGTV propagation method (as this study used BHK-21 cells) or the age of the mice (we used relatively younger mice aged 6–7 weeks).

To visualize the dynamics of TBF infection routes more effectively, we developed a reporter virus system for in vivo imaging by following a strategy similar to that described by Tsetsarkin et al. (26). However, we observed a tendency for the Nluc reporter gene to be lost after viral rescue (Fig. 1B). This loss primarily stems from homologous recombination occurring between the C68 and the complete C gene sequences (Fig. 1A). Because the reporter virus is significantly attenuated compared to the WT virus, the reporter virus would be out-competed by the WT virus when original virus pool contained some WT virus. To obtain a stable reporter virus free of WT virus contamination, we performed seven rounds of plaque purification (36). It has been previously documented that mutations can occur in 5′ cyclization sequences post-plaque purification, potentially compromising the stability of the West Nile reporter virus (37). Interestingly, our reporter LGTV demonstrated genomic stability in vitro, with no nucleotide substitutions in the Nluc reporter gene up to P10. We sequenced two C genes, cyclization sequences, and kiss loops which may influence the homologous recombination of the virus. Yet, we did not identify any mutations in these regions (data not shown), suggesting that mutations may arise in other regions of the viral genome, thereby enhancing the stability of the viral genome. To corroborate this hypothesis, it will be necessary to sequence the viral entire genome and create single mutated viruses to evaluate the viral stability in future studies. Despite these findings, it is important to note that the Nluc reporter LGTV was significantly attenuated in comparison to the WT-LGTV both in vitro and in vivo. Notably, we observed that the insertion of reporter genes could increase the length of the viral genome, which may perturb genome cyclization or viral RNA packaging, thereby affecting the viral life cycle. This phenomenon is commonly observed in other reporter flaviviruses (38 –41). Nonetheless, the properties of the Nluc reporter LGTV facilitated its excellent performance in vivo and ex vivo, providing valuable insights into the dynamics of TBF infection routes.

Further, we used the Nluc-LGTV reporter virus to monitor gastrointestinal tract infection by TBF in mice. The bioluminescence of TBF infection was visualized in vivo, with distinct Nluc expression observed in the gastrointestinal tract, potentially reflecting the tissue tropism of TBF in vivo. In addition, high luciferase activity was observed in several organs. However, the brain surprisingly exhibited low luminescence intensity despite high viral RNA loads, which could be due to the low permeability of the substrate through the blood-brain barrier (27). This finding was similar to the real-time dynamic tissue distribution of other previously reported flavivirus infections (40, 42). Additionally, dam-to-pup transmission of TBF was also investigated in dams infected after parturition. We detected distinct luciferase activity in two of the three litters during breastfeeding. However, not all pups were infected, possibly because of the reduced virulence of the Nluc reported virus compared to the WT virus. Dams would recover from Nluc-LGTV infection, and the viral doses that some pups received from dams’ milk did not cause the infection. However, some limitations could be present in the reporter virus system, as frequent anesthesia and substrate injections can affect the survival of pups, and we were unable to monitor the infection dynamics daily.

Finally, a recent study on the transmission of enteric viruses through saliva demonstrated that the saliva of infected infants could directly transmit enteric viruses to their mothers' mammary glands through backflow during suckling (43). Moreover, LGTV RNA has also been detected in mouse saliva (35), suggesting that future research should investigate the transmission of TBF infections via saliva from infants to mothers during breastfeeding.

In summary, our study provided comprehensive evidence of breast milk transmission of TBF in mice and has generated a Nluc-LGTV reporter virus system, which is a valuable tool for studying TBF transmission routes. Although our findings highlight the importance of educating the public on the transmission risks associated with unpasteurized dairy products, further studies on breast milk transmission of TBEV are needed.

MATERIALS AND METHODS

Animal and ethics statement

A129 mice lacking type I interferon (IFN-α/β) receptors were generously provided by Gengfu Xiao from the Wuhan Institute of Virology, Chinese Academy of Sciences.

All animal experiments adhered to the institutional guidelines for animal research and were approved by the Administration of Affairs Concerning Experimental Animals of the People’s Republic of China. Prior to conducting the animal experiments, our protocols were reviewed and approved by the Ethics Committee of the Animal House facility of the Wuhan Institute of Virology, Chinese Academy of Sciences (Permit Number: WIVA07202004).

Cells and viruses

Baby hamster kidney cells (BHK-21; CCL-10; ATCC) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS) (Life Technology, Australia), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultivation was maintained in 5% CO₂ at 37°C.

The parental wild-type LGTV and Nluc-LGTV viruses were recovered from infectious clone (IC)-transfected BHK-21 cells.

Construction of infectious clones

To generate the wild-type TP21 strain of LGTV (GenBank accession no. AF253419) (44) infectious clone, four fragments containing the complete viral genome of LGTV were synthesized (Sangon Biotech) and cloned into the low-copy-number plasmid pACYC177 by enzyme ligation. These fragments include (i) fragment 1, containing 1–2,314 nt of the LGTV genome, overlapped with a cytomegalovirus (CMV) promoter and cloned into pACYC177 at the KpnI and AvrII sites; (ii) fragment 2, containing 2,315–4,980 nt of the genome, was cloned into pACYC177 at AvrII and KasI sites, with the beta-globin intron from pACYC177-ZIKV-FL (45) inserted at 2494 nt of the genome; (iii) fragment 3, containing 4,981–8,566 of the genome, was cloned into pACYC177 at KasI and ClaI sites; and (iv) in fragment 4, containing 8,567–11,076 of the genome, the hepatitis D virus ribozyme (HDVr) sequence-simian virus 40 (SV40) poly(A), which was directly amplified from pACYC177-ZIKV-FL, were fused and cloned into pACYC177 at CalI and XhoI sites.

The infectious clone of Nluc-LGTV was generated using the pACYC177-LGTV backbone. The Nluc-2A, amplified from the pNL1.1 Nluc vector (Promega), overlapped with CMV-5′UTR-C68 and C-prM-E450. Then ORF-shifting mutations with a single A nucleotide insertion at C8 and a G nucleotide deletion at C68 were introduced in C68. Finally, CMV-5′UTR-C68-Nluc-2A-C-prM-E450 was cloned into pACYC177-LGTV at the SnaBI and AvrII sites.

Virus recovery and stock production

Infectious virus rescue experiments were conducted as described by Li et al. (25). Briefly, BHK-21 cells were plated onto 35 mm culture dishes and cultured at 37°C with 5% CO₂ for 24 h. After the BHK-21 cells reached 85% confluence, they were transfected with 2 µg per dish of LGTV and Nluc-LGTV ICs using Lipofectamine 3000 (Life Technologies). The supernatant was then harvested at 4 days post-transfection, clarified by centrifugation, labeled as P0, and stored at −80°C. For LGTV stocks, the titrated P0 virus was used to infect fresh BHK-21 cells at an MOI of 0.01 for 72 h to generate P1 stocks. The P1 viral stocks were subsequently aliquoted and stored at −80°C until ready for use. The Nluc-LGTV stocks were collected after seven rounds of plaque purification and then aliquoted and stored at −80°C until ready for use.

Plaque assay

The Plaque assay was conducted according to a previously described method (25). Briefly, BHK-21 cells were grown to 85% confluence in 24-well plates and incubated with viral samples (200 µL for all samples except mouse milk samples, which were incubated with 150 µL) diluted 10-fold in DMEM. After an incubation period of 1.5 h, the cells were overlaid with 1.25% methylcellulose containing 2% FBS. Following a 4-day incubation period, the cells were fixed with 4% paraformaldehyde for 2 h and stained with a 0.5% crystal violet solution. Plaque morphology and number were recorded, and virus titrations were expressed as PFU/mL.

Plaque purification

The double-plaque assay was performed for plaque purification as previously described (40). BHK-21 cells in six-well plates were grown to 80% confluence and inoculated with 500 µL of 10-fold serial dilutions of viral samples in DMEM. After a 1.5-h incubation period, 3 mL of 0.6% agarose supplemented with 2% FBS was added to each well. Following a 4-day incubation period, 3 mL of agarose containing 0.33% neutral red was added to each well, and the plaques were photographed or picked after incubation for another 24 h.

Virus growth kinetics

Confluent monolayers of BHK-21 cells were infected with LGTV, Nluc-LGTV (MOI of 0.01), or mock-infected in triplicate with 10⁵ cells per well in 12-well plates. After 1.5 h of virus adsorption at 37°C, the cells were washed with chilled PBS, overlaid with 1 mL of DMEM containing 2% FBS, and incubated at 37°C. At various time points (i.e., 12, 24, 48, 72, and 96 hpi), viral titers were determined by plaque assay. The presence of Nluc from LGTV- or Nluc-LGTV-infected cells was quantified using the Nano-Glo Luciferase Assay System (Promega), according to the manufacturer’s instructions.

Reverse transcription-PCR

RNA from virus-infected BHK-21 cells (MOI of 0.01) was extracted using TRIzol LS reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. The viral genome spanning nucleotides 108–417 was amplified using a pair of primers (forward primer 5′-GCTTAGGAGAACAAGAGCTGGG-3′ and reverse primer 5′-CTTCTACTTCCTCGGCGGTG-3′) with PrimeScript RT reagent kit (Takara) and KOD-Plus-Neo (TOYOBO). The amplified DNA products were separated on a 1% agarose gel.

Quantitative reverse transcription PCR

Total RNA was extracted from serum, organs, or stomach milk clots using the TRIzol reagent (Thermo Fisher Scientific) and reverse transcribed with the PrimeScript RT reagent kit (Takara). A pair of primers (forward primer 5′-CCCACCTGGAAAACAGAGACT-3′ and reverse primer 5′-TGCAGGGCTCTCTTGGTAGAT-3′) was used to amplify the region spanning positions 981–1,132 of the LGTV genome. All RT-qPCR assays were performed with SYBR green master mix (Bio-Rad) on a CFX96 touch real-time PCR detection system (Bio-Rad). The cycling conditions were as follows: 95°C for 3 min, followed by 39 cycles of 95°C for 10 s, 55°C for 10 s, and 65°C for 30 s. pACYC177-LGTV was used as the standard plasmid, and log dilutions of the DNA standard were included in each assay. The virus concentration was determined by interpolation onto a curve comprising 10-fold serial dilutions of the standards.

Immunofluorescence assay

BHK-21 cells were mock-infected or infected (MOI of 1) with LGTV or Nluc-LGTV. At 72 h post-infection, the cells were fixed with 4% paraformaldehyde at 4°C overnight, permeabilized using 0.3% Triton X-100 in PBS for 15 min at room temperature, incubated overnight with TBEV E protein hyperimmune rabbit serum (1:1,000) at 4°C overnight, washed with PBS three times, and stained with a goat anti-rabbit IgG, Alexa Fluor 555 (CST, 1:1,000) at 37°C for 30 min. After washing three times with PBS, cell nuclei were stained with Hoechst 33258, and fluorescent signal images were taken using a confocal microscope (A1 HD25, Nikon).

Transmission electron microscopy

BHK-21 cells infected with LGTV or Nluc-LGTV and mouse tissues were pre-fixed with 2.5% glutaraldehyde at 4°C overnight. After washing three times in PBS, the samples were post-fixed with 1% osmium tetroxide. Afterward, the samples were dehydrated in acetone and then re-embedded and sectioned. Ultra-thin sections (100 nm) were examined using a transmission electron microscope (G2 20 TWIN, Tecnai).

Animal experiments

A129 mice at 6–7 weeks old were anesthetized with isoflurane following gaseous sedation and infected with 10³, 10², or 10¹ PFU WT-LGTV or Nluc-LGTV via intraperitoneal (IP) injection. The mock-infected mice were injected with PBS via the same route. After infection, the mice were monitored daily for morbidity (body weight) and mortality (survival rate). Mice that lost more than 25% of their initial body weight were considered to have reached the experimental endpoint and were euthanized, and the survival curves were plotted using the Kaplan-Meier method (46). The 50% lethal dose (LD₅₀) for each LGTV was determined using the Reed and Muench method (47).

For different infection routes of LGTV, A129 mice at 6–7 weeks old were anesthetized with isoflurane following gaseous sedation and infected with 10⁵ PFU WT-LGTV or Nluc-LGTV via IG, IP, or FP. After infection, the mice were monitored daily for morbidity (body weight) and mortality (survival rate). Mice that lost more than 25% of their initial body weight were considered to have reached the experimental endpoint and were euthanized, and the survival curves were plotted using the Kaplan-Meier method. The hearts, livers, spleens, lungs, kidneys, brains, testes, uteri, and intestines of the infected mice were removed, weighed, and homogenized with zirconia beads in 1 mL of PBS and stored at −80°C until use.

Milk samples

To collect fresh mouse milk, A129 dams (15–20 weeks old) were intraperitoneally inoculated with 100 µL LGTV (1 × 10⁵ PFU) or 100 µL PBS 2 days post-parturition. Dams and pups were separated for 2 h prior to milking. Before milk collection, each dam received 0.1 mL (2 IU) of oxytocin intraperitoneally. The teat’s base was then gently stimulated to let the milk down from the teat. Mouse milk was collected with a pipette and stored at −80°C until use (48, 49).

For SMC isolation, dams (15–20 weeks old) were intraperitoneally inoculated with 100 µL LGTV (1 × 10⁵ PFU) or 100 µL PBS 2 days post-parturition. SMCs were removed from the pup’s stomachs and lysed in 1 mL TRIzol reagent (Thermo Fisher Scientific) for 5 min at room temperature.

Bioluminescence imaging

Nluc-LGTV-infected or uninfected mice were shaved in advance and anesthetized via subcutaneous injection of avertin (150 µL/10 g of 2.5% solution). Nano-Glo substrate (Promega) was diluted 1:20 in PBS, and each mouse was intraperitoneally injected with 100 µL of the mixture (25 µL per pup). Bioluminescence data were collected using an IVIS CCD camera system (PerkinElmer) and further processed using Living Image software (version 4.4, PerkinElmer). For ex vivo imaging of organs, Nluc-LGTV-infected and uninfected mice were euthanized 5 dpi after substrate injection, and the heart, liver, spleen, lung, kidney, brain, testis, uterus, mammary gland, and gastrointestinal tract were isolated for imaging.

Histology and immunostaining

Tissues were fixed in 4% paraformaldehyde overnight. Paraffin embedding and sectioning were performed as previously described (40) and stained with H&E. For immunohistochemical staining, after heat-mediated antigen retrieval, the sections were stained with rabbit anti-E protein domain III (EDIII) of the LGTV (1:1000). While for immunofluorescence staining, the following antibodies were used: Krt14 (Abcam, 1:150), Krt8 (Abcam, 1:100), and LGTV EDIII (1:1,000). Images were captured using a whole-slide digital panoramic scanner (3D-Histech).

Statistical analysis

GraphPad Prism 7 was used for graphical and statistical analyses. All data are presented as mean ± SD or mean ± SEM for each group. A P value <0.05 was considered statistically significant.

ACKNOWLEDGMENTS

The authors thank Prof Fei Deng of the National Virus Resource Center (NVRC) for providing the TBEV E protein hyperimmune rabbit serum. The authors thank Xuefang An, Fan Zhang, He Zhao, Yuzhou Xiao, and Li Li from the Laboratory Animal Centerof Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS), for their help in animal experiments. The authors also thank Ding Gao, Pei Zhang, Anna Du, and Juan Min of the Core Facility and Technical of WIV, CAS, for their support with confocal microscopy and transmission electron microscopy. Our sincere appreciation also goes to Figdraw (www.figdraw.com) for assistance in creating Fig. 5A and 5E.

This work was supported by the National Natural Science Foundation of China (NSFC, No. 82272329), the National Key Research and Development Project (No. 2022YFC2302700), and Hubei Province Laboratory Animal Resource Development and Utilization Project (No. 2020DFE023).

Y.M., Z.Z., and Y.Z. designed research; Y.M., Y.Z., and T.W. performed research; W.Z., and T.W. contributed new reagents/analytic tools; Y.M., Z.Z., N.Z., W.Z., and W.Y. analyzed data; and Y.M., Z.Z., and W.Z. wrote the paper.

Contributor Information

Wanpo Zhang, Email: zwp@mail.hzau.edu.cn.

Zhenhua Zheng, Email: zhengzh@wh.iov.cn.

Mark T. Heise, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.01709-23.

Supplemental figures. jvi.01709-23-s0001.pdf.

Fig. S1 to S3.

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DOI: 10.1128/jvi.01709-23.SuF1

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REFERENCES

1. Lindquist L, Vapalahti O. 2008. Tick-borne encephalitis. Lancet 371:1861–1871. doi: 10.1016/S0140-6736(08)60800-4 [DOI] [PubMed] [Google Scholar]
2. Golovljova I, Vene S, Sjölander KB, Vasilenko V, Plyusnin A, Lundkvist A. 2004. Characterization of tick-borne encephalitis virus from Estonia. J Med Virol 74:580–588. doi: 10.1002/jmv.20224 [DOI] [PubMed] [Google Scholar]
3. Buczek AM, Buczek W, Buczek A, Wysokińska-Miszczuk J. 2022. Food-borne transmission of tick-borne encephalitis virus-spread, consequences, and prophylaxis. Int J Environ Res Public Health 19:1812. doi: 10.3390/ijerph19031812 [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Beauté J, Spiteri G, Warns-Petit E, Zeller H. 2018. Tick-borne encephalitis in Europe, 2012 to 2016. Euro Surveill 23:1800201. doi: 10.2807/1560-7917.ES.2018.23.45.1800201 [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Jääskeläinen AE, Tikkakoski T, Uzcátegui NY, Alekseev AN, Vaheri A, Vapalahti O. 2006. Siberian subtype tickborne encephalitis virus, Finland. Emerg Infect Dis 12:1568–1571. doi: 10.3201/eid1210.060320 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Brockmann SO, Oehme R, Buckenmaier T, Beer M, Jeffery-Smith A, Spannenkrebs M, Haag-Milz S, Wagner-Wiening C, Schlegel C, Fritz J, Zange S, Bestehorn M, Lindau A, Hoffmann D, Tiberi S, Mackenstedt U, Dobler G. 2018. A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016. Euro Surveill 23:17-00336. doi: 10.2807/1560-7917.ES.2018.23.15.17-00336 [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Markovinović L, Kosanović Ličina ML, Tešić V, Vojvodić D, Vladušić Lucić I, Kniewald T, Vukas T, Kutleša M, Krajinović LC. 2016. An outbreak of tick-borne encephalitis associated with raw goat milk and cheese consumption, Croatia, 2015. Infection 44:661–665. doi: 10.1007/s15010-016-0917-8 [DOI] [PubMed] [Google Scholar]
8. Paulsen KM, Stuen S, das Neves CG, Suhel F, Gurung D, Soleng A, Stiasny K, Vikse R, Andreassen ÅK, Granquist EG. 2019. Tick-borne encephalitis virus in cows and unpasteurized cow milk from Norway. Zoonoses Public Health 66:216–222. doi: 10.1111/zph.12554 [DOI] [PubMed] [Google Scholar]
9. Caini S, Szomor K, Ferenczi E, Szekelyne Gaspar A, Csohan A, Krisztalovics K, Molnar Z, Horvath J. 2012. Tick-borne encephalitis transmitted by unpasteurised cow milk in western Hungary, September to October 2011. Euro Surveill 17:20128. [PubMed] [Google Scholar]
10. Balogh Z, Ferenczi E, Szeles K, Stefanoff P, Gut W, Szomor KN, Takacs M, Berencsi G. 2010. Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk. J Virol Methods 163:481–485. doi: 10.1016/j.jviromet.2009.10.003 [DOI] [PubMed] [Google Scholar]
11. Holzmann H, Aberle SW, Stiasny K, Werner P, Mischak A, Zainer B, Netzer M, Koppi S, Bechter E, Heinz FX. 2009. Tick-borne encephalitis from eating goat cheese in a mountain region of Austria. Emerg Infect Dis 15:1671–1673. doi: 10.3201/eid1510.090743 [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Kerlik J, Avdičová M, Štefkovičová M, Tarkovská V, Pántiková Valachová M, Molčányi T, Mezencev R. 2018. Slovakia reports highest occurrence of alimentary tick-borne encephalitis in Europe: analysis of tick-borne encephalitis outbreaks in Slovakia during 2007-2016. Travel Med Infect Dis 26:37–42. doi: 10.1016/j.tmaid.2018.07.001 [DOI] [PubMed] [Google Scholar]
13. Van Tongeren HA. 1955. Encephalitis in Austria. IV. Excretion of virus by milk of the experimentally infected goat. Arch Gesamte Virusforsch 6:158–162. [PubMed] [Google Scholar]
14. Ličková M, Fumačová Havlíková S, Sláviková M, Klempa B. 2021. Alimentary infections by tick-borne encephalitis virus. Viruses 14:56. doi: 10.3390/v14010056 [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Gritsun TS, Lashkevich VA, Gould EA. 2003. Tick-borne encephalitis. Antiviral Res 57:129–146. doi: 10.1016/s0166-3542(02)00206-1 [DOI] [PubMed] [Google Scholar]
16. Yu C, Achazi K, Möller L, Schulzke JD, Niedrig M, Bücker R, Wang T. 2014. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers. PLoS One 9:e96957. doi: 10.1371/journal.pone.0096957 [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Balogh Z, Egyed L, Ferenczi E, Bán E, Szomor KN, Takács M, Berencsi G. 2012. Experimental infection of goats with tick-borne encephalitis virus and the possibilities to prevent virus transmission by raw goat milk. Intervirology 55:194–200. doi: 10.1159/000324023 [DOI] [PubMed] [Google Scholar]
18. Kenyon RH, Rippy MK, McKee KT, Zack PM, Peters CJ. 1992. Infection of Macaca radiata with viruses of the tick-borne encephalitis group. Microb Pathog 13:399–409. doi: 10.1016/0882-4010(92)90083-z [DOI] [PubMed] [Google Scholar]
19. Mercer J, Helenius A. 2009. Virus entry by macropinocytosis. Nat Cell Biol 11:510–520. doi: 10.1038/ncb0509-510 [DOI] [PubMed] [Google Scholar]
20. Mann TZ, Haddad LB, Williams TR, Hills SL, Read JS, Dee DL, Dziuban EJ, Pérez-Padilla J, Jamieson DJ, Honein MA, Shapiro-Mendoza CK. 2018. Breast milk transmission of flaviviruses in the context of Zika virus: a systematic review. Paediatr Perinat Epidemiol 32:358–368. doi: 10.1111/ppe.12478 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Kerlik J, Avdičová M, Musilová M, Bérešová J, Mezencev R. 2022. Breast milk as route of tick-borne encephalitis virus transmission from mother to infant. Emerg Infect Dis 28:1060–1061. doi: 10.3201/eid2805.212457 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Weber E, Finsterbusch K, Lindquist R, Nair S, Lienenklaus S, Gekara NO, Janik D, Weiss S, Kalinke U, Överby AK, Kröger A. 2014. Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses. J Virol 88:12202–12212. doi: 10.1128/JVI.01215-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Pletnev AG, Men R. 1998. Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. Proc Natl Acad Sci U S A 95:1746–1751. doi: 10.1073/pnas.95.4.1746 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Su Y, Walker JR, Park Y, Smith TP, Liu LX, Hall MP, Labanieh L, Hurst R, Wang DC, Encell LP, Kim N, Zhang F, Kay MA, Casey KM, Majzner RG, Cochran JR, Mackall CL, Kirkland TA, Lin MZ. 2020. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nat Methods 17:852–860. doi: 10.1038/s41592-020-0889-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Li P, Yao C, Wang T, Wu T, Yi W, Zheng Y, Miao Y, Sun J, Tan Z, Liu Y, Zhang X, Wang H, Zheng Z. 2021. Recovery of a far-eastern strain of tick-borne encephalitis virus with a full-length infectious cDNA clone. Virol Sin 36:1375–1386. doi: 10.1007/s12250-021-00396-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Tsetsarkin KA, Liu G, Shen K, Pletnev AG. 2016. Kissing-loop interaction between 5' and 3' ends of tick-borne Langat virus genome 'bridges the gap' between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development. Nucleic Acids Res 44:3330–3350. doi: 10.1093/nar/gkw061 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Ago K, Nagoshi N, Imaizumi K, Kitagawa T, Kawai M, Kajikawa K, Shibata R, Kamata Y, Kojima K, Shinozaki M, Kondo T, Iwano S, Miyawaki A, Ohtsuka M, Bito H, Kobayashi K, Shibata S, Shindo T, Kohyama J, Matsumoto M, Nakamura M, Okano H. 2022. A non-invasive system to monitor in vivo neural graft activity after spinal cord injury. Commun Biol 5:803. doi: 10.1038/s42003-022-03736-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Li C-C, Shapiro H, Tsiobikas C, Selfors LM, Chen H, Rosenbluth J, Moore K, Gupta KP, Gray GK, Oren Y, Steinbaugh MJ, Guerriero JL, Pinello L, Regev A, Brugge JS. 2020. Aging-associated alterations in mammary epithelia and stroma revealed by single-cell RNA sequencing. Cell Rep 33:108566. doi: 10.1016/j.celrep.2020.108566 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Offerdahl DK, Clancy NG, Bloom ME. 2016. Stability of a tick-borne flavivirus in milk. Front Bioeng Biotechnol 4:40. doi: 10.3389/fbioe.2016.00040 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Regla-Nava JA, Viramontes KM, Vozdolska T, Huynh AT, Villani T, Gardner G, Johnson M, Ferro PJ, Shresta S, Kim K. 2019. Detection of Zika virus in mouse mammary gland and breast milk. PLoS Negl Trop Dis 13:e0007080. doi: 10.1371/journal.pntd.0007080 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Pang W, Lin Y-L, Xin R, Chen X-X, Lu Y, Zheng C-B, Yang L-M, Zheng Y-T. 2021. Zika virus transmission via breast milk in suckling mice. Clin Microbiol Infect 27:469. doi: 10.1016/j.cmi.2020.04.021 [DOI] [PubMed] [Google Scholar]
32. Hubert M, Chiche A, Legros V, Jeannin P, Montange T, Gessain A, Ceccaldi PE, Vidy A. 2019. Productive infection of mouse mammary glands and human mammary epithelial cells by Zika virus. Viruses 11:950. doi: 10.3390/v11100950 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Pogodina VV. 1960. An experimental study on the pathogenesis of tick-borne encephalitis following alimentary infection. Part 2. A study of the methods of excretion of viruses from the body of the white mouse. Vopr Virusol 5:279–285. [PubMed] [Google Scholar]
34. Levkovich EN, Pogodina VV. 1958. Infection through the alimentary tract with tick-borne encephalitis. Vopr Virusol 3:145–150. [PubMed] [Google Scholar]
35. Schreier S, Cebulski K, Kröger A. 2021. Contact-dependent transmission of Langat and tick-borne encephalitis virus in type I interferon receptor-1 deficient mice. J Virol 95:e02039-20. doi: 10.1128/JVI.02039-20 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Jia F, Zhu X, Xu F. 2016. A single adaptive point mutation in Japanese encephalitis virus capsid is sufficient to render the virus as a stable vector for gene delivery. Virology 490:109–118. doi: 10.1016/j.virol.2016.01.001 [DOI] [PubMed] [Google Scholar]
37. Zhang P-T, Shan C, Li X-D, Liu S-Q, Deng C-L, Ye H-Q, Shang B-D, Shi P-Y, Lv M, Shen B-F, Qin C-F, Zhang B. 2016. Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay. Virus Res 211:17–24. doi: 10.1016/j.virusres.2015.09.015 [DOI] [PubMed] [Google Scholar]
38. Baker C, Shi PY. 2020. Construction of stable reporter flaviviruses and their applications. Viruses 12:1082. doi: 10.3390/v12101082 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Baker C, Xie X, Zou J, Muruato A, Fink K, Shi PY. 2020. Using recombination-dependent lethal mutations to stabilize reporter flaviviruses for rapid serodiagnosis and drug discovery. EBioMedicine 57:102838. doi: 10.1016/j.ebiom.2020.102838 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Wang T, Li P, Zhang Y, Liu Y, Tan Z, Sun J, Ke X, Miao Y, Luo D, Hu Q, Xu F, Wang H, Zheng Z. 2020. In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy. Theranostics 10:6430–6447. doi: 10.7150/thno.43177 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Samsa MM, Mondotte JA, Caramelo JJ, Gamarnik AV. 2012. Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation. J Virol 86:1046–1058. doi: 10.1128/JVI.05431-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Li XF, Li XD, Deng CL, Dong HL, Zhang QY, Ye Q, Ye HQ, Huang XY, Deng YQ, Zhang B, Qin CF. 2017. Visualization of a neurotropic flavivirus infection in mouse reveals unique viscerotropism controlled by host type I interferon signaling. Theranostics 7:912–925. doi: 10.7150/thno.16615 [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Ghosh S, Kumar M, Santiana M, Mishra A, Zhang M, Labayo H, Chibly AM, Nakamura H, Tanaka T, Henderson W, Lewis E, Voss O, Su Y, Belkaid Y, Chiorini JA, Hoffman MP, Altan-Bonnet N. 2022. Enteric viruses replicate in salivary glands and infect through saliva. Nature 607:345–350. doi: 10.1038/s41586-022-04895-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Campbell MS, Pletnev AG. 2000. Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence. Virology 269:225–237. doi: 10.1006/viro.2000.0220 [DOI] [PubMed] [Google Scholar]
45. Li P, Ke X, Wang T, Tan Z, Luo D, Miao Y, Sun J, Zhang Y, Liu Y, Hu Q, Xu F, Wang H, Zheng Z. 2018. Zika virus attenuation by codon pair deoptimization induces sterilizing immunity in mouse models. J Virol 92:e00701-18. doi: 10.1128/JVI.00701-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Efron B. 1988. Logistic regression, survival analysis, and the Kaplan-Meier curve. J Am Stat Assoc 83:414–425. doi: 10.1080/01621459.1988.10478612 [DOI] [Google Scholar]
47. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493–497. doi: 10.1093/oxfordjournals.aje.a118408 [DOI] [Google Scholar]
48. Muranishi Y, Parry L, Averous J, Terrisse A, Maurin AC, Chaveroux C, Mesclon F, Carraro V, Bruhat A, Fafournoux P, Jousse C. 2016. Method for collecting mouse milk without exogenous oxytocin stimulation. Biotechniques 60:47–49. doi: 10.2144/000114373 [DOI] [PubMed] [Google Scholar]
49. DePeters EJ, Hovey RC. 2009. Methods for collecting milk from mice. J Mammary Gland Biol Neoplasia 14:397–400. doi: 10.1007/s10911-009-9158-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Supplemental figures. jvi.01709-23-s0001.pdf.

Fig. S1 to S3.

jvi.01709-23-s0001.pdf^{(1.2MB, pdf)}

DOI: 10.1128/jvi.01709-23.SuF1

[B1] 1. Lindquist L, Vapalahti O. 2008. Tick-borne encephalitis. Lancet 371:1861–1871. doi: 10.1016/S0140-6736(08)60800-4 [DOI] [PubMed] [Google Scholar]

[B2] 2. Golovljova I, Vene S, Sjölander KB, Vasilenko V, Plyusnin A, Lundkvist A. 2004. Characterization of tick-borne encephalitis virus from Estonia. J Med Virol 74:580–588. doi: 10.1002/jmv.20224 [DOI] [PubMed] [Google Scholar]

[B3] 3. Buczek AM, Buczek W, Buczek A, Wysokińska-Miszczuk J. 2022. Food-borne transmission of tick-borne encephalitis virus-spread, consequences, and prophylaxis. Int J Environ Res Public Health 19:1812. doi: 10.3390/ijerph19031812 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Beauté J, Spiteri G, Warns-Petit E, Zeller H. 2018. Tick-borne encephalitis in Europe, 2012 to 2016. Euro Surveill 23:1800201. doi: 10.2807/1560-7917.ES.2018.23.45.1800201 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Jääskeläinen AE, Tikkakoski T, Uzcátegui NY, Alekseev AN, Vaheri A, Vapalahti O. 2006. Siberian subtype tickborne encephalitis virus, Finland. Emerg Infect Dis 12:1568–1571. doi: 10.3201/eid1210.060320 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Brockmann SO, Oehme R, Buckenmaier T, Beer M, Jeffery-Smith A, Spannenkrebs M, Haag-Milz S, Wagner-Wiening C, Schlegel C, Fritz J, Zange S, Bestehorn M, Lindau A, Hoffmann D, Tiberi S, Mackenstedt U, Dobler G. 2018. A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016. Euro Surveill 23:17-00336. doi: 10.2807/1560-7917.ES.2018.23.15.17-00336 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Markovinović L, Kosanović Ličina ML, Tešić V, Vojvodić D, Vladušić Lucić I, Kniewald T, Vukas T, Kutleša M, Krajinović LC. 2016. An outbreak of tick-borne encephalitis associated with raw goat milk and cheese consumption, Croatia, 2015. Infection 44:661–665. doi: 10.1007/s15010-016-0917-8 [DOI] [PubMed] [Google Scholar]

[B8] 8. Paulsen KM, Stuen S, das Neves CG, Suhel F, Gurung D, Soleng A, Stiasny K, Vikse R, Andreassen ÅK, Granquist EG. 2019. Tick-borne encephalitis virus in cows and unpasteurized cow milk from Norway. Zoonoses Public Health 66:216–222. doi: 10.1111/zph.12554 [DOI] [PubMed] [Google Scholar]

[B9] 9. Caini S, Szomor K, Ferenczi E, Szekelyne Gaspar A, Csohan A, Krisztalovics K, Molnar Z, Horvath J. 2012. Tick-borne encephalitis transmitted by unpasteurised cow milk in western Hungary, September to October 2011. Euro Surveill 17:20128. [PubMed] [Google Scholar]

[B10] 10. Balogh Z, Ferenczi E, Szeles K, Stefanoff P, Gut W, Szomor KN, Takacs M, Berencsi G. 2010. Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk. J Virol Methods 163:481–485. doi: 10.1016/j.jviromet.2009.10.003 [DOI] [PubMed] [Google Scholar]

[B11] 11. Holzmann H, Aberle SW, Stiasny K, Werner P, Mischak A, Zainer B, Netzer M, Koppi S, Bechter E, Heinz FX. 2009. Tick-borne encephalitis from eating goat cheese in a mountain region of Austria. Emerg Infect Dis 15:1671–1673. doi: 10.3201/eid1510.090743 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Kerlik J, Avdičová M, Štefkovičová M, Tarkovská V, Pántiková Valachová M, Molčányi T, Mezencev R. 2018. Slovakia reports highest occurrence of alimentary tick-borne encephalitis in Europe: analysis of tick-borne encephalitis outbreaks in Slovakia during 2007-2016. Travel Med Infect Dis 26:37–42. doi: 10.1016/j.tmaid.2018.07.001 [DOI] [PubMed] [Google Scholar]

[B13] 13. Van Tongeren HA. 1955. Encephalitis in Austria. IV. Excretion of virus by milk of the experimentally infected goat. Arch Gesamte Virusforsch 6:158–162. [PubMed] [Google Scholar]

[B14] 14. Ličková M, Fumačová Havlíková S, Sláviková M, Klempa B. 2021. Alimentary infections by tick-borne encephalitis virus. Viruses 14:56. doi: 10.3390/v14010056 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Gritsun TS, Lashkevich VA, Gould EA. 2003. Tick-borne encephalitis. Antiviral Res 57:129–146. doi: 10.1016/s0166-3542(02)00206-1 [DOI] [PubMed] [Google Scholar]

[B16] 16. Yu C, Achazi K, Möller L, Schulzke JD, Niedrig M, Bücker R, Wang T. 2014. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers. PLoS One 9:e96957. doi: 10.1371/journal.pone.0096957 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Balogh Z, Egyed L, Ferenczi E, Bán E, Szomor KN, Takács M, Berencsi G. 2012. Experimental infection of goats with tick-borne encephalitis virus and the possibilities to prevent virus transmission by raw goat milk. Intervirology 55:194–200. doi: 10.1159/000324023 [DOI] [PubMed] [Google Scholar]

[B18] 18. Kenyon RH, Rippy MK, McKee KT, Zack PM, Peters CJ. 1992. Infection of Macaca radiata with viruses of the tick-borne encephalitis group. Microb Pathog 13:399–409. doi: 10.1016/0882-4010(92)90083-z [DOI] [PubMed] [Google Scholar]

[B19] 19. Mercer J, Helenius A. 2009. Virus entry by macropinocytosis. Nat Cell Biol 11:510–520. doi: 10.1038/ncb0509-510 [DOI] [PubMed] [Google Scholar]

[B20] 20. Mann TZ, Haddad LB, Williams TR, Hills SL, Read JS, Dee DL, Dziuban EJ, Pérez-Padilla J, Jamieson DJ, Honein MA, Shapiro-Mendoza CK. 2018. Breast milk transmission of flaviviruses in the context of Zika virus: a systematic review. Paediatr Perinat Epidemiol 32:358–368. doi: 10.1111/ppe.12478 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Kerlik J, Avdičová M, Musilová M, Bérešová J, Mezencev R. 2022. Breast milk as route of tick-borne encephalitis virus transmission from mother to infant. Emerg Infect Dis 28:1060–1061. doi: 10.3201/eid2805.212457 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Weber E, Finsterbusch K, Lindquist R, Nair S, Lienenklaus S, Gekara NO, Janik D, Weiss S, Kalinke U, Överby AK, Kröger A. 2014. Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses. J Virol 88:12202–12212. doi: 10.1128/JVI.01215-14 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Pletnev AG, Men R. 1998. Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4. Proc Natl Acad Sci U S A 95:1746–1751. doi: 10.1073/pnas.95.4.1746 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Su Y, Walker JR, Park Y, Smith TP, Liu LX, Hall MP, Labanieh L, Hurst R, Wang DC, Encell LP, Kim N, Zhang F, Kay MA, Casey KM, Majzner RG, Cochran JR, Mackall CL, Kirkland TA, Lin MZ. 2020. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nat Methods 17:852–860. doi: 10.1038/s41592-020-0889-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Li P, Yao C, Wang T, Wu T, Yi W, Zheng Y, Miao Y, Sun J, Tan Z, Liu Y, Zhang X, Wang H, Zheng Z. 2021. Recovery of a far-eastern strain of tick-borne encephalitis virus with a full-length infectious cDNA clone. Virol Sin 36:1375–1386. doi: 10.1007/s12250-021-00396-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Tsetsarkin KA, Liu G, Shen K, Pletnev AG. 2016. Kissing-loop interaction between 5' and 3' ends of tick-borne Langat virus genome 'bridges the gap' between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development. Nucleic Acids Res 44:3330–3350. doi: 10.1093/nar/gkw061 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Ago K, Nagoshi N, Imaizumi K, Kitagawa T, Kawai M, Kajikawa K, Shibata R, Kamata Y, Kojima K, Shinozaki M, Kondo T, Iwano S, Miyawaki A, Ohtsuka M, Bito H, Kobayashi K, Shibata S, Shindo T, Kohyama J, Matsumoto M, Nakamura M, Okano H. 2022. A non-invasive system to monitor in vivo neural graft activity after spinal cord injury. Commun Biol 5:803. doi: 10.1038/s42003-022-03736-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Li C-C, Shapiro H, Tsiobikas C, Selfors LM, Chen H, Rosenbluth J, Moore K, Gupta KP, Gray GK, Oren Y, Steinbaugh MJ, Guerriero JL, Pinello L, Regev A, Brugge JS. 2020. Aging-associated alterations in mammary epithelia and stroma revealed by single-cell RNA sequencing. Cell Rep 33:108566. doi: 10.1016/j.celrep.2020.108566 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Offerdahl DK, Clancy NG, Bloom ME. 2016. Stability of a tick-borne flavivirus in milk. Front Bioeng Biotechnol 4:40. doi: 10.3389/fbioe.2016.00040 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Regla-Nava JA, Viramontes KM, Vozdolska T, Huynh AT, Villani T, Gardner G, Johnson M, Ferro PJ, Shresta S, Kim K. 2019. Detection of Zika virus in mouse mammary gland and breast milk. PLoS Negl Trop Dis 13:e0007080. doi: 10.1371/journal.pntd.0007080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Pang W, Lin Y-L, Xin R, Chen X-X, Lu Y, Zheng C-B, Yang L-M, Zheng Y-T. 2021. Zika virus transmission via breast milk in suckling mice. Clin Microbiol Infect 27:469. doi: 10.1016/j.cmi.2020.04.021 [DOI] [PubMed] [Google Scholar]

[B32] 32. Hubert M, Chiche A, Legros V, Jeannin P, Montange T, Gessain A, Ceccaldi PE, Vidy A. 2019. Productive infection of mouse mammary glands and human mammary epithelial cells by Zika virus. Viruses 11:950. doi: 10.3390/v11100950 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Pogodina VV. 1960. An experimental study on the pathogenesis of tick-borne encephalitis following alimentary infection. Part 2. A study of the methods of excretion of viruses from the body of the white mouse. Vopr Virusol 5:279–285. [PubMed] [Google Scholar]

[B34] 34. Levkovich EN, Pogodina VV. 1958. Infection through the alimentary tract with tick-borne encephalitis. Vopr Virusol 3:145–150. [PubMed] [Google Scholar]

[B35] 35. Schreier S, Cebulski K, Kröger A. 2021. Contact-dependent transmission of Langat and tick-borne encephalitis virus in type I interferon receptor-1 deficient mice. J Virol 95:e02039-20. doi: 10.1128/JVI.02039-20 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Jia F, Zhu X, Xu F. 2016. A single adaptive point mutation in Japanese encephalitis virus capsid is sufficient to render the virus as a stable vector for gene delivery. Virology 490:109–118. doi: 10.1016/j.virol.2016.01.001 [DOI] [PubMed] [Google Scholar]

[B37] 37. Zhang P-T, Shan C, Li X-D, Liu S-Q, Deng C-L, Ye H-Q, Shang B-D, Shi P-Y, Lv M, Shen B-F, Qin C-F, Zhang B. 2016. Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay. Virus Res 211:17–24. doi: 10.1016/j.virusres.2015.09.015 [DOI] [PubMed] [Google Scholar]

[B38] 38. Baker C, Shi PY. 2020. Construction of stable reporter flaviviruses and their applications. Viruses 12:1082. doi: 10.3390/v12101082 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Baker C, Xie X, Zou J, Muruato A, Fink K, Shi PY. 2020. Using recombination-dependent lethal mutations to stabilize reporter flaviviruses for rapid serodiagnosis and drug discovery. EBioMedicine 57:102838. doi: 10.1016/j.ebiom.2020.102838 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Wang T, Li P, Zhang Y, Liu Y, Tan Z, Sun J, Ke X, Miao Y, Luo D, Hu Q, Xu F, Wang H, Zheng Z. 2020. In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy. Theranostics 10:6430–6447. doi: 10.7150/thno.43177 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Samsa MM, Mondotte JA, Caramelo JJ, Gamarnik AV. 2012. Uncoupling cis-Acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation. J Virol 86:1046–1058. doi: 10.1128/JVI.05431-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Li XF, Li XD, Deng CL, Dong HL, Zhang QY, Ye Q, Ye HQ, Huang XY, Deng YQ, Zhang B, Qin CF. 2017. Visualization of a neurotropic flavivirus infection in mouse reveals unique viscerotropism controlled by host type I interferon signaling. Theranostics 7:912–925. doi: 10.7150/thno.16615 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43. Ghosh S, Kumar M, Santiana M, Mishra A, Zhang M, Labayo H, Chibly AM, Nakamura H, Tanaka T, Henderson W, Lewis E, Voss O, Su Y, Belkaid Y, Chiorini JA, Hoffman MP, Altan-Bonnet N. 2022. Enteric viruses replicate in salivary glands and infect through saliva. Nature 607:345–350. doi: 10.1038/s41586-022-04895-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44. Campbell MS, Pletnev AG. 2000. Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence. Virology 269:225–237. doi: 10.1006/viro.2000.0220 [DOI] [PubMed] [Google Scholar]

[B45] 45. Li P, Ke X, Wang T, Tan Z, Luo D, Miao Y, Sun J, Zhang Y, Liu Y, Hu Q, Xu F, Wang H, Zheng Z. 2018. Zika virus attenuation by codon pair deoptimization induces sterilizing immunity in mouse models. J Virol 92:e00701-18. doi: 10.1128/JVI.00701-18 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46. Efron B. 1988. Logistic regression, survival analysis, and the Kaplan-Meier curve. J Am Stat Assoc 83:414–425. doi: 10.1080/01621459.1988.10478612 [DOI] [Google Scholar]

[B47] 47. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493–497. doi: 10.1093/oxfordjournals.aje.a118408 [DOI] [Google Scholar]

[B48] 48. Muranishi Y, Parry L, Averous J, Terrisse A, Maurin AC, Chaveroux C, Mesclon F, Carraro V, Bruhat A, Fafournoux P, Jousse C. 2016. Method for collecting mouse milk without exogenous oxytocin stimulation. Biotechniques 60:47–49. doi: 10.2144/000114373 [DOI] [PubMed] [Google Scholar]

[B49] 49. DePeters EJ, Hovey RC. 2009. Methods for collecting milk from mice. J Mammary Gland Biol Neoplasia 14:397–400. doi: 10.1007/s10911-009-9158-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice

Yuanjiu Miao

Yue Zheng

Ting Wang

Wenfu Yi

Nailou Zhang

Wanpo Zhang

Zhenhua Zheng

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

RESULTS

Generation of Nluc-LGTV reporter virus

Fig 1.

In vivo characterization of Nluc-LGTV

Fig 2.

LGTV infection in mice via the intragastric route

Fig 3.

Infectious LGTV present in mice milk and mammary glands

Fig 4.

Dam-to-pup infection of LGTV through breastfeeding

Fig 5.

LGTV infects epithelial cells of the mammary gland

Fig 6.

DISCUSSION

MATERIALS AND METHODS

Animal and ethics statement

Cells and viruses

Construction of infectious clones

Virus recovery and stock production

Plaque assay

Plaque purification

Virus growth kinetics

Reverse transcription-PCR

Quantitative reverse transcription PCR

Immunofluorescence assay

Transmission electron microscopy

Animal experiments

Milk samples

Bioluminescence imaging

Histology and immunostaining

Statistical analysis

ACKNOWLEDGMENTS

Contributor Information

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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