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Establishment and application of a wild neonatal mouse model infected with an Echovirus 30 isolate

Virology Journal volume 22, Article number: 69 (2025) Cite this article

Abstract

Background

Echovirus 30 (E30) is a significant pathogen associated with various illnesses such as viral meningitis, viral myocarditis. Currently, there are no specific drugs or vaccines targeting this virus. An appropriate animal model is imperative for assessing drug and vaccine efficacy.

Methods

This investigation aimed to establish a neonatal mouse model using a clinical isolate E30/A538 and apply it to screen anti-E30 drugs. The study involved evaluating the susceptibility of different mouse strains to the isolate, determining the infectious dose, transmission route, and optimal age of the mice. This model was then used to assess antiviral efficacy.

Results

Neonatal ICR mice infected intracranially with 5LD50 of E30/A538 at one-day-old displayed clinical symptoms such as tremors, lethargy, limb paralysis, and mortality. Importantly, the E30/A538-infected mice exhibited brain neuron apoptosis and severe myocardial necrolysis, closely resembling human infections. Elevated levels of viral RNA and positive antigen presence were predominantly detected in the brains and hearts of infected mice. Using this model to assess antiviral efficacy, it was demonstrated that interferon-α2a inhibited E30/A538 replication in vivo, mitigated histopathological changes in the brain, spinal cord, and myocardium, and enhanced the survival rate of neonatal mice.

Conclusions

In summary, this research established a wild neonatal mouse model of E30/A538 isolate infection that mirrors the characteristics of human infection. The model demonstrated the efficacy of interferon-α2a in combating E30. This model would serve as a foundation for investigating the pathogenesis of E30, as well as for assessing the efficacy of vaccines and other antiviral treatments against E30.

Introduction

Echovirus 30 (E30) is classified under species B of the Enteroviruses (EVs) genus within the Picornaviridae family. Its genome consists of a positive single-stranded RNA approximately 7400 nucleotides long, containing an open reading frame (ORF) responsible for encoding viral structural and non-structural proteins [1,2,3]. Recent years have witnessed localized outbreaks of E30 in various countries, including the United States, France, Italy, Brazil, India, South Korea, and several EU/EEA countries [3,4,5,6]. Additionally, multiple outbreaks of E30 have been reported in different regions of China, such as Shandong, Jiangsu, Gansu, and Zhejiang [7,8,9]. Due to underreporting and differences in diagnostic capabilities, the global incidence and mortality rates of E30 have not been fully documented. The rise in cases linked to E30 co-infection with other EV-B viruses has resulted in the emergence of recombinant virus strains with modified transmissibility or pathogenicity, posing challenges to prevention and control efforts [6, 10, 11].

E30 is a significant pathogen responsible for viral meningitis (VM) and viral encephalitis (VE), as well as other illnesses such as hand-foot-mouth disease, acute flaccid paralysis (AFP), and viral myocarditis (VMC). The population affected by Echovirus 30 infection is primarily concentrated in the 0–15 age group, with a particularly high infection rate among infants and young children aged 0–4 [7, 12, 13]. Currently, there are no specific medications or preventive vaccines available for this pathogen. The utilization of animal models infected with the virus is crucial for studying its pathogenesis and for the development of new drugs and vaccines [14,15,16]. Several animal models for EV have been successfully established, with the mouse model being widely preferred due to its cost-effectiveness, ease of maintenance, short experimental duration, stable population systems, reliability, and accurate replication of symptoms of central nervous system diseases [17,18,19,20,21]. While weak strains may initially have low susceptibility to mice, they can mutate to enhance their virulence after continuous passage in mice, thus establishing a mouse model for highly adaptive strains. This process inevitably leads to changes in the pathogenic mechanism and pathological damage of the virus strains [22]. If clinical isolates are capable of infecting mice and inducing typical symptoms, it can provide valuable insights into the pathogenicity, pathogenic process, and pathogenic mechanism of the clinical isolates in humans. This knowledge is essential for the development and application of vaccines and drugs for human use. Therefore, the development of a cost-effective and stable mouse model infected with an E30 isolate that replicates the clinical characteristics of human infection is imperative [23].

This research sought to develop a stable mouse model infected with a clinical isolate of E30. The neonatal mouse model displayed symptoms resembling those seen in humans, including encephalitis and myocarditis. To evaluate the applicability of the model, we applied it to analyze the antiviral properties of rhIFN-α2a and rhIFN-γ. The establishment of this model offers a valuable resource for conducting fundamental investigations into E30 and for assessing the effectiveness of particular medications and preventative vaccines.

Materials and methods

Ethics statement

The study utilized ICR, BALB/c, KM, and C57BL/6 mice obtained from SJA Laboratory Animal Corporation in China, which were free of specific pathogens. The experimental animals were licensed under SCXK (Hunan) 2019-0004. All animal procedures were conducted in compliance with the guidelines approved by the Ethics Committee of Laboratory Animal Management at Guilin Medical College (Number: GLMC202303003).

Cells, virus strain and antibody

Human rhabdomyosarcoma cells (RD) obtained from Sangon Biotech in China were maintained in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum from Invitrogen in the USA. The E30/A538 isolate (A538/GD/CHN/2009, GenBank accession number: ON129560) was sourced from fecal samples of an individual diagnosed with acute flaccid paralysis, subsequently amplified using universal primers, and characterized through genomic sequencing and phylogenetic analysis. The clinical isolate E30/A538 was identified as the F genotype by constructing a phylogenetic tree based on the E30 VP1 gene (Fig. S1). The E30 VP1 polyclonal antibody used in this study was prepared in our laboratory by immunizing rabbits with the E30 VP1 protein expressed in E. coli. Blood samples were collected from the rabbits, and the serum was separated and purified using affinity chromatography.

Plaque assay

The plaque assay was conducted utilizing 12-well plates that housed RD cell monolayers. The cells were exposed to incremental tenfold dilutions of E30/A538 for 2 h at 37℃. After rinsing, the media in the dish were replaced with DMEM-1% low melting point agarose (Beyotime, ST107) supplemented with 2% FBS. Plaques were generated through incubation for a period of 5 days at 37℃. The cells were then stained with 0.01% neutral red, and the resulting plaques were documented photographically. In the process of plaque purification, 5 to 10 plaques were selected, and two additional rounds of plaque purification were performed, respectively. Strains triggering early CPE onset were selected and subjected to two rounds of amplification in RD cells before being preserved as viral stocks.

Western blot

Cultures of E30/A538-infected cells at various time points post-infection (12, 24, 48, and 72 h) were freeze-thawed 3 times and centrifuged to remove cellular debris. The obtained supernatants were subjected to boiling for 10 min at 100 °C in a loading buffer comprising 50 mM Tris, 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 1% β-mercaptoethanol. Subsequently, viral proteins were separated through 10% SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Following blocking with 3% BSA, the membranes were exposed to mouse sera targeting the virus (dilution 1:2,000) for 2 h, and then to a horseradish peroxidase-conjugated secondary antibody for 1 h. β-actin was used as a loading control. Protein visualization was achieved using a commercial ECL kit (EpiZyme, China), and band intensity was quantified utilizing Image Lab software (Bio-Rad, USA).

TCID50 assay

A monolayer of RD cells was infected with a 10-fold gradient dilution of E30/A538 in a 96-well plate and subsequently incubated at 37 ℃ in a 5% CO2 environment for a period of 5–7 days to monitor the cytopathic effect (CPE). Wells with most cells exhibiting obvious CPE are considered positive. The viral titer was determined utilizing the Spearman Karber method, employing the formula logTCID50 /100µL = Xm 1/2d-d(∑pi /100), where Xm represents the logarithm of the highest concentration dilution of the virus utilized, and d signifies the logarithm of the dilution coefficient (multiple), while Σ Pi denotes the cumulative percentage of cellular lesions observed at each dilution level. The viral stock titer was quantified as 109.375 TCID50/mL.

Animal experiments

In the model building process, neonatal mice were randomly divided into groups of six each. The sensitivity comparison of different mice strain to E30/A538 was firstly conducted by challenging one-day-old ICR, BALB/c, KM, and C57BL/6 mice with 102.208 TCID50/mL of E30/A538, 20 µL per mouse via the IC route with 1 ml insulin injection needle. ICR mice were ultimately selected as the animal strain for the model construction. In order to measure an appropriate challenge dose, one-day-old ICR mice were exposed to E30/A538 ranging from 103.676 TCID50 to 101.676 TCID50 per mouse through the intracerebral (IC) route. After conducting three replicate experiments, it was verified that the minimum lethal dose was 5 LD50, equivalent to 102.208 TCID50. The optimal age for infection was determined by administering 5LD50 of E30/A538 via the IC route to neonatal mice at different ages (1, 3, 5, and 7 days). The most suitable infection route was identified by challenging one-day-old neonatal mice with 5LD50 of E30/A538 per mouse through IC, intraperitoneal (IP), or intramuscular (IM) routes. In the application of the established model to the evaluation of anti-E30/A538 effects of IFN, the neonatal mice were randomly divided into groups of twelve each. Either IFN-γ (200U) or IFN-α2a (200U) was administered through the IP route at 1-hour post-infection. From days 1 to 6 dpi, equivalent quantities of IFN were administered daily. All negative control (NC) mice were administered an equivalent volume of sterile 1× PBS. All experiments included appropriate negative controls, such that the only variable between the NC groups in the strain experiments was the mouse strain. Observations on body weight changes, clinical symptoms, and clinical scores were made daily until 21 dpi, and survival rates were calculated. The clinical scores were based on the following criteria [24]: Score 0 (Healthy): Mice exhibit normal activity levels, foraging behavior, and interaction with their environment, with no signs of discomfort. Score 1 (Lethargy and reduced activity): Mice show reduced activity, lack of interaction with their surroundings, and may display sluggish movement or remain in one place for extended periods. Score 2 (Weight loss or hind limb weakness): Mice exhibit significant weight loss, reduced muscle tone, or obvious hind limb weakness that affects their normal mobility or balance. Score 3 (Unilateral hind limb paralysis): One hind limb of the mouse is completely paralyzed and cannot be used for movement or support. This can be confirmed by testing the limb’s responsiveness. Score 4 (Bilateral hind limb paralysis): Both hind limbs of the mouse are completely paralyzed, rendering the mouse unable to walk. Assistance is required for mobility. During observation, gentle handling can confirm complete loss of leg movement. Score 5 (Death): The mouse is unresponsive, showing no signs of life, and cannot be revived.

Histopathological examination and immunohistochemical analysis

One-day-old ICR neonatal mice were challenged through the IC route with 5LD50 of E30/A538 or PBS. When the mice in the virus group reached a score exceeding 4, euthanasia was performed on all the mice. Tissues were collected, processed, embedded in paraffin, and sectioned at 5-µm thickness. Hematoxylin and eosin staining were performed, followed by histopathological examination by an experienced clinical pathologist. Immunohistochemical staining for viral antigen detection in various tissues was carried out using the MaxVision TMHRP-Polymer Anti-Mouse IHC kit from MXB Biotechnologies, China. The primary antibody utilized in the immunohistochemistry was the E30 polyclonal mouse antibody (dilution 1:1500) developed in-house.

Viral RNA quantification

For cell samples, RD cells were infected with the E30/A538 strain at a concentration of 106.375 TCID50 per well in 6-well plates. The method for obtaining the supernatant from the culture is shown in Western blot. The collection of mouse tissue samples is shown in Histopathological examination and immunohistochemical analysis. Subsequently, the total viral RNA from virus-infected RD cells or mouse tissues was extracted using TRIzol reagent from Solarbio, China. The viral load of E30/A538 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR) with a specific primer pair for the VP1 gene (5’-ACTTCTCTGTGCGGCTGCTG-3’, 5’-CACTGGCTACTGTGTCGGCTAC-3’) and a standard curve equation (Y=-4.0507X + 47.929, R² = 0.999). This standard curve was generated using a reference plasmid containing the E30 VP1 fragment inserted into the pMD-19T vector. The amplification conditions are as follows: initial denaturation at 94 °C for 5 min. Denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 2 min (35 cycles). Analyze the melting curve from 60 °C to 95 °C to confirm specificity.

Statistical analysis

All experiments were repeated at least three times. Data processing and statistical analysis were conducted utilizing GraphPad Prism 9.0 software. The Mantel-Cox log-rank test was employed to assess variations in the survival rate of suckling mice across different groups within 21 dpi. Unpaired t-tests were utilized to examine changes in body weight and clinical scores between the virus and NC groups within the same 21-day period. Furthermore, Dunnett’s multiple comparisons test was applied to evaluate changes in body weight, clinical scores, and CPE inhibition rates among the various groups.

Results

Plaque purification and characterization of the E30/A538 isolate

Following the infection of RD cells with the E30/A538 isolate, the supernatant from infected cells cultured for 72 h was subjected to a 10-fold concentration gradient dilution and utilized to infect RD cell monolayers. Subsequently, cells were covered with an agarose-containing medium overlay. After 5 days, distinct plaques were observed at dilutions of 10− 3 and 10− 4 (Fig. 1A). The virus was isolated through monoclonal selection via plaque assay and subsequently employed in cellular and animal studies. After infecting RD cells with E30/A538, noticeable CPE was observed 24 h after infection, and by the 72-hour time point, almost all cells had detached (Fig. 1B). Utilizing a western blot assay, the cell lysate supernatant was probed using anti-E30 virus serum as the primary antibody. This analysis revealed the presence of VP1 proteins of E30 at molecular weights of 35 kDa (Fig. 1C). Furthermore, the qPCR method was employed to quantify viral copies. It was observed that a gradual increase in virus titer in the cell culture supernatant post 12 h of infection, peaking at 48 h (Fig. 1D).

Fig. 1
figure 1

The major characterizations of the E30/A538 isolate. RD cell monolayers were inoculated with E30/A538 and subsequently covered with an agarose-containing medium overlay. After 5 days, plaques were visualized by staining with neutral red (A). The CPE of E30/A538 on RD cells was observed at 12, 24, 48, and 72 h after infection using an inverted microscope (B). The cell lysate supernatant was analyzed by western blot (C) and qPCR (D).

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The E30/A538 isolate exhibited strong susceptibility in ICR, BALB/c, and KM mice

ICR, BALB/c, KM and C57BL/6 mice were infected with E30/A538 via IC. ICR, BALB/c and KM exhibited clear clinical manifestations, including weight loss, arched back, quadriplegia (Fig. 2D to G), and had a mortality rate of 100% (Fig. 2C). The three indicators (body weight, clinical score, and survival rate) showed no significant difference among them (Fig. 2A to C). In contrast, C57BL/6 mice primarily exhibited thinness (Fig. 2G) and a poor mental state, with a mortality rate of 33.3% and a clinical score of 1.67 (Fig. 2A to C). All NC mice of these strains were gradually gained weight (Fig. S2A), clinically symptom-free (Fig. S2B), and survived (Fig. S2C) during a 21-day observation period. These results indicated that E30/A538 exhibited strong susceptibility in ICR, BALB/c, and KM mice, but poor susceptibility in C57BL/6 mice. ICR mice were eventually chosen to establish E30/A538 infection animal models due to their strong reproductive capacity.

Fig. 2
figure 2

Susceptibility of different strains of mice to E30/A538. ICR, BALB/c, KM, and C57BL/6 mice were infected through the IC route with the same dose of E30/A538. The body weights (A), clinical scores (B), and survival rates (C) of each group of suckling mice (n = 6) were recorded until 21 days post-infection (dpi). Black arrows marked the quadriplegia (D) in ICR mice on the 5d post-infection, the moribund state (E) of BALB/c mice on the 5d post-infection, the arched back (F) of KM mice on the 5d post-infection, and the weight loss (G) of in C57BL/6 mice on the 12d post-infection. The results displayed the mean ± standard error of the mean (SEM). ****P < 0.0001, ***P < 0.001, “ns” indicates not significant versus ICR

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Establishment of an E30/A538-infected neonatal ICR mouse model

The investigation into dosing revealed that the presentation of infected mice to E30/A538 in terms of weight, health score, and survival was dependent on the dosage administered (Fig. 3A to C). Mice were exposed to E30/A538 ranging from 103.676 TCID50 to 101.676 TCID50. The results showed that the survival rate was 33.3% in mice received 101.676 TCID50 and 0% in the other two groups (Fig. 3C). To facilitate a more detailed observation of post-infection symptoms in suckling mice and assess the drug’s antiviral effectiveness in vivo, 5LD50 (equivalent to 102.208 TCID50) was the optimal infection dose for establishing a mouse model. Results from the age screening indicated that all one-day-old suckling mice perished within 12 dpi. Additionally, the mortality rate of 3-day-old mice within 21 dpi was recorded at 50%, with some mice displaying a gradual alleviation of clinical symptoms that eventually disappeared. Notably, five- and seven-day-old mice exhibited no susceptibility to the E30/A538 challenge, with all individuals surviving in a healthy condition within 21 dpi (Fig. 3D to F). The susceptibility of ICR mice to E30/A538 decreased with advancing age, prompting the selection of one-day-old suckling mice for subsequent investigations due to their uniform mortality post-disease onset. These neonatal mice were exposed to 5LD50 of E30/A538 per mouse through IC, IP, or IM administration routes. All three routes resulted in disease manifestation in the suckling mice, with mortality rates of 100%, 83.33%, and 83.33% within 21 days post-infection (Fig. 3I), and corresponding clinical scores of 5.00, 4.17, and 4.17, respectively (Fig. 3H). Notably, mice infected via the IC route exhibited more severe clinical symptoms (Fig. S3A) and pathological changes (Fig. S3B) compared to the other routes, leading to the selection of the intracranial route for subsequent experiments. Consistent results were obtained through repeated experiments. The gradual decline in body weight observed in nursing mice starting from 4 dpi onwards (Fig. 4A). During the period spanning days 5 to 10 dpi, the mice exhibited signs of fatigue, hunched posture, tremors, complete loss of limb function, ultimately leading to death (Fig. 4D). By day 21, there was a complete mortality rate of 100% (Fig. 4C), accompanied by a clinical severity score of 5 (Fig. 4B).

Fig. 3
figure 3

The E30/A538-infected ICR mouse model was developed by optimizing three factors: infection dose, route, and mouse age. Body weights (A, D, and G), clinical scores (B, E, and H), and survival rates (C, F, and I) were calculated for each group (n = 6). The NC was injected with sterile 1× PBS. The results displayed were the mean ± SEM. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. “ns” means “not significant” compared to the NC group

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Fig. 4
figure 4

The repeatability of the E30/A538-infected suckling mouse model. The weight changes (A), clinical scores (B), survival rates (C), and clinical manifestations (D) of the mice were recorded until 21 dpi, with a sample size of n = 6. Black arrows indicate the signs of limb paralysis in mice at 5dpi. The results displayed the mean ± SEM. ****P < 0.0001, ***P < 0.001

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Infectious characteristics in different tissues of ICR suckling mice infected with E30/A538

The histopathological examination of different tissues using hematoxylin and eosin (HE) staining revealed pathological alterations in the brain, heart, spinal cord, liver, skeletal muscle, and back-fat pad of infected suckling mice (Fig. 5A; Table 1). Compared to control mice, infected mice displayed distinctive pathological features in various tissues. For instance, the brain tissue exhibited foam-like cells, nuclear fission, focal sclerosing necrosis, and scattered lymphocytes. Similarly, myocardial tissue showed signs of necrosis, disintegration, and edema. Hepatocytes displayed diffuse lesions characterized by degeneration and necrosis, along with inflammatory cell infiltration and fibrous tissue hyperplasia. The spinal cord exhibited focal sclerosing necrosis. Immunohistochemical (IHC) analysis revealed the presence of brown-yellow antigen particles in all mentioned tissues above except for the liver (Fig. 5B; Table 1). Notably, the brain and heart of infected mice displayed more severe pathological changes and higher antigen expressions compared to other tissues. Viral RNA was detected in various tissues of E30/A538-infected suckling mice through quantitative real-time polymerase chain reaction (qRT-PCR), with tissues showing strong antigen expression correlating with higher viral loads. The brain tissue exhibited the highest viral load at 104 copies/mg, while the mean viral loads in the heart, and dorsal fat pad were 9.55 × 103, and 5.78 × 103 copies/mg, respectively (Fig. 5C; Table 1). Their viral load was significantly higher than that of other tissues, suggesting that these tissues might serve as the primary target organs for viral replication. Notably, while E30 VP1 gene copies were detectable in the liver, no antigen was identified in the liver through HI, which could be attributed to the liver’s rich blood supply and the presence of viremia.

Fig. 5
figure 5

Histological, immunohistochemical, and viral load analyses in various tissues of ICR suckling mice infected with E30/A538. Tissues were harvested on the 7th day post-infection, at which time the clinical scores for the E30 group and the NC group were 5 and 0, respectively. (A) Tissues from each group were stained with hematoxylin and eosin (H&E). Scale bar represents 100 µm. “6×” represents the image of the small black box has been magnified 6 times. Yellow arrows marked the lesions in E30/A538-infected neonatal mice. (B) Immunohistochemical staining of each group. Scale bar represents 100 µm. “6×” represents the image of the small black box has been magnified 6 times. The brain and heart contained a large number of brownish-yellow E30 antigen particles. (C) The average viral load in tissue of suckling mice infected with E30/A538. The viral loads in the tissues of mice (n = 3) with clinical scores of 4 or higher were measured using qRT-PCR

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Table 1 Infection characteristics in various tissues of ICR suckling mice infected with E30
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Evaluation of the anti-E30/A538 effect of IFN in the neonatal mouse model

In our preliminary study, IFN-α2a and IFN-γ were showed that they could effectively inhibit E30 replication in vitro (Fig. S4). Therefore, in this study, we further utilized the E30/A538-infected ICR neonatal model to evaluate the efficacy of IFN-α2a and IFN-γ against E30 in vivo, as depicted in Fig. 6. In comparison to the control group, mice treated with IFN-α2a + E30 exhibited a notable increase in body weight and a reduction in clinical symptoms (Fig. 6B and E). However, mice treated with IFN-γ + E30 still displayed severe clinical manifestations. The survival rates of mice in the IFN-α2a + E30, IFN-γ + E30, and E30 groups at 21 days post-infection were 66.67%, 0%, and 0%, respectively (Fig. 6D), with corresponding clinical scores of 2.17, 5, and 5 (Fig. 6C). The mice treated with IFN-α2a + E30 exhibited different clinical scores, with 42% scoring 0, 25% scoring 2, and 33% scoring 5. Overall, mice treated with IFN-α2a + E30 revealed less severe lesions compared to those in the E30/A538 model group by histopathological analysis (Fig. 6F). IFN-α2a treatment reduced the necrotic area in the heart, mitigated inflammatory responses in the brain and spinal cord, resolved mild liver tissue edema (Fig. 6F; Table 2), and increased the likelihood of survival. These findings suggest that the E30/A538-infected ICR mouse model is suitable for assessing the efficacy of antiviral drugs in vivo.

Fig. 6
figure 6

Anti-E30/A538 effect of IFN-α2a and IFN-γ in vivo. (A) Schematic diagram of antiviral experiments: Twelve one-day-old ICR mice were infected with 5LD50 E30/A538 by IC. IFN-α2a and IFN-γ were injected at 1 hpi, and the same dose was re-administered every 24 hpi for the next 6 days. The NC were injected with sterile 1×PBS. (B) Clinical manifestations: At 11 dpi, all the mice in the virus group died, while those in the IFN-α2a treatment group experienced weight loss. The mice in the IFN-α2a control group and NC group remained healthy. (C-E) The body weights (C), clinical scores (D), and survival rates (E) of each group of suckling mice were recorded until 21 dpi. The results displayed the mean ± S.E.M. ***P < 0.001; “ns” indicates no significant difference compared to the E30/A538 group. (F) IFN-α2a reduced the extent of pathological tissue changes. The brain, heart, spinal cord, limb muscles, fat, and liver of each group were stained with HE. Scale bar represents 100 µm.“6×” represents the image of the small black box has been magnified 6 times. Yellow arrows marked the specific pathological changes are shown in the Table 2

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Table 2 Characteristics of different tissues infection in E30/A538-infected ICR suckling mice treated with IFN
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Discussion

In recent times, there has been a significant rise in cases of viral meningitis in China due to the E30 virus. However, there remains a dearth of appropriate animal models for E30, hindering further research into its pathogenic mechanisms and potential antiviral treatments. Various neonatal mouse models have been successfully developed for studying other viruses such as EVA71, CVA16, CVA10, CVA6, and CVB5, enabling investigations into vaccine and drug candidates as well as disease pathogenesis [17, 25,26,27,28]. In our preliminary experiments, we selected three E30 clinical isolates (P4/ZS/CHN/2009, GenBank: MN018207; P8/ZS/CHN/2009, GenBank: MN018208.1; A538/GD/CHN/2009, GenBank: ON129560.1) to infect mice. The virulence and pathogenicity of these isolates varied. Among them, E30/A538 exhibited strong virulence, leading to the rapid death of neonatal mice (Fig. S5). This makes it more suitable for constructing an E30 infection animal model. ICR mice were selected for their robust reproductive capacity and susceptibility to enteroviruses, making them a genetically uniform cohort. Traditionally, constructing animal models for enterovirus infection involves first adapting the virus within the mice and then increasing its titer in vitro to establish a stock for infecting neonatal mice. However, the E30/A538 clinical isolate used in this study exhibited strong sensitivity to ICR mice. This means that it can infect and consistently cause lethal outcomes in these mice without requiring adaptation to create a mouse-adapted strain. Through optimization of factors such as infection dosage, route of infection, and age of the animals, a stable neonatal mouse model infected with E30/A538 was successfully established using 1-day-old ICR mice.

During the model construction process, to minimize the possibility of infection or injury resulting from human handling, we conducted injections using a 1 mL insulin syringe under sterile conditions and included both negative and drug control groups for comparison. The subsequent analysis of E30/A538-infected mice involved a detailed observation of the neurological symptoms induced by the virus, including manifestations such as thinning, tremors, lethargy, bending, and limb paralysis. Previous studies have highlighted the utility of animal models in exploring the mechanisms underlying enterovirus infections. Notably, a recent study introduced an IFNAR-/- mice model infected with E30, which exhibited significant pathological alterations primarily in skeletal muscle and brain tissues [29]. IFNAR-/- mice, lacking type I interferon (IFN) receptors, are more vulnerable to viral infections due to their compromised innate immunity [30]. Consequently, the histopathological features observed in gene knockout mice utilized as models for viral infections may not accurately reflect the clinicopathological characteristics seen in individuals with intact immune systems during viral infections, rendering them unsuitable for assessing the effectiveness of antiviral drugs and vaccines. In contrast, the E30/A538-infected ICR mouse model developed in this investigation demonstrated viral presence in all tissues, with notably higher viral concentrations detected in the brain and heart. During tissue analysis, we used clinical scores as the determining criterion. Tissues were collected from E30-infected mice when their clinical score was greater than or equal to 4, along with samples from negative control, drug control, and drug-treated mice for pathological analysis. Histopathological analysis revealed brain edema, brain neuron apoptosis, and lymphocyte infiltration in infected mice. Furthermore, severe myocardial necrolysis, inflammatory cell infiltration in the spinal cord, and scattered lymphocytes in limb muscles were also observed.

Since the blood-brain barrier in neonatal mice is not fully developed, the virus can spread systemically via viremia after intracranial infection, replicating in target organs and producing lesions similar to those caused by oral or respiratory routes of infection [31]. Currently, many studies on neonatal mouse models of enterovirus infection have chosen to use intracranial infection to establish the model [23, 26, 32, 33]. In our study, the virus could directly affect muscles or disrupt neuromuscular junctions, interfering with the transmission of motor signals. Key findings indicate that infected mice exhibit direct neuropathological damage, with the possibility that the virus may target motor neurons. Lesions in the brain and other tissues observed after intracranial infection mirror those reported in other studies using oral or respiratory routes of infection, suggesting that the pathogenic features are comparable. Additionally, The HE and IHC results in this study indicate that the E30 virus exhibits tropism for cardiac tissue and can cause myocardial damage. The virus may infect cardiomyocytes directly through hematogenous spread or indirectly affect the heart via neural pathways, disrupting the autonomic nervous system and impairing cardiac function. Myocardial infection can lead to myocarditis, cardiomyocyte damage, and even heart failure. These findings were consistent with the clinicopathological features observed in human patients, such as meningitis, myocarditis, and acute flaccid paralysis, suggesting a potential link between these complications resulting from E30 infection, an exacerbated immune response, and elevated cytokine expression triggered by the virus invasion and subsequent immune reactions. This research conducted repeated infections on suckling mice to confirm the stability of the model and observed consistent infection characteristics leading to mortality, demonstrating the model’s high operability and reproducibility.

Subsequently, the study evaluated the in vivo anti-E30 effects of rhIFN-α2a and rhIFN-γ using this model. Interferons (IFNs) are a group of cytokines with immunomodulatory, antitumor, and antiviral properties [34]. They recognize various pathogens through pattern recognition receptors (PRRs) on cells, inducing the synthesis of antiviral proteins. IFNs play a crucial role in vertebrate defense against pathogen invasion. The research revealed that rhIFN-γ did not exhibit significant antiviral effects in vivo. Instead, it expedited the progression of the disease (Fig. 6D and E). Studies have indicated that the elevated IFN-γ, causing an imbalanced IFN-γ/IL-4 ratio, was a significant factor in severe disease development [35]. IFN-γ participates in the immune response against enteroviruses through multiple mechanisms, including activating immune cells, regulating the expression of antiviral proteins, inhibiting viral replication, and enhancing cytotoxic responses. However, enteroviruses may evade the antiviral effects of IFN-γ through various mechanisms. Studies have shown that enteroviruses, such as EV-A71, rely on their proteases 2 A and 3D to interfere with IFN-γ activity via different pathways. Specifically, 2 A inhibits STAT1 phosphorylation, while 3D suppresses IFN-γ protein expression [36]. This suggests that the antiviral role of IFN-γ against enteroviruses may be limited by viral evasion strategies. Such mechanisms could potentially explain the poor efficacy of IFN-γ against E30 observed in this study. Further research is needed to elucidate the specific mechanisms involved. Additionally, the study demonstrated that the high concentration of rhIFN-α2a showed no animal toxicity and effectively inhibited viral replication in suckling mice. It also reduced myocardial injury, decreased inflammatory cytokine levels in the brain, spinal cord, and limb muscles, eliminated liver edema, and notably enhanced survival rates. Studies have shown that IFN-α2a exerts its antiviral effects by activating antiviral proteins such as PKR and OAS, thereby inhibiting viral replication. This may be the potential mechanism underlying its therapeutic effect against E30 infection [37]. An existing research has also proved rhIFN-α2a’s antiviral properties against Coxsackievirus A4 (CVA4), suggesting the potential broad-spectrum anti- enteroviruses activity of rhIFN-α2a [19].

Conclusion

In summary, an E30/A538 isolate was employed to establish a lethal E30 neonatal mouse model with a high degree of stability and replicability. This model exhibited characteristic manifestations of E30 infection, akin to those witnessed in human cases of the disease. This study employed the model to demonstrated the efficacy of IFN-α2a in combating E30, highlighting its potential importance in the clinical management of viral meningitis and associated complications induced by E30. Moreover, it would serve as a reliable research tool for evaluating the effectiveness of different pharmaceuticals or vaccines in future studies.

Data availability

No datasets were generated or analysed during the current study.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81660280; No. 31760262; No. 32360191), Guangxi Medical and Health Key Discipline Construction Project, and Project for Enhancing Young and Middle-aged Teacher’s Research Basis Ability in Colleges of Guangxi (No.2022KY0488).

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Author notes

  1. Ying Qu, Jing Wang and Yongbei Chen contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, The Second Affiliated Hospital of Guilin Medical University, Guilin, China

    Ying Qu, Jing Wang, Yongbei Chen, Ning Zhang, Qiliang Liu & Hongbo Liu

  2. College of Pharmacy, Guilin Medical University, Guilin, Guangxi, China

    Ying Qu & Jing Wang

  3. Guangxi Key Laboratory of Metabolic Reprogramming and Intelligent Medical Engineering for Chronic Diseases, Guangxi Clinical Research Center for Diabetes and Metabolic Diseases, Department of Laboratory Medicine, The Second Affiliated Hospital of Guilin Medical University, Guilin, China

    Yongbei Chen, Ning Zhang & Hongbo Liu

  4. Department of Pathology, The Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi, China

    Shengjun Xiao

  5. College of Laboratory Medicine, Guilin Medical University, Guilin, Guangxi, China

    Yunyi He & Hongbo Liu

  6. Guangdong Provincial Institute of Public Health, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, Guangdong, China

    Huanying Zheng

  7. College of Intelligent Medicine and Biotechnology, Guilin Medical University, Guilin, Guangxi, China

    Qiliang Liu

  8. Key Laboratory of Medical Biotechnology and Translational Medicine, Education Department of Guangxi Zhuang Autonomous, Nanning, China

    Qiliang Liu

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Contributions

Y.Q.: Performed experiments, Software and Data Curation, Analyzed the data, Writing– Original Draft. J.W.: Performed experiments, Software and Data Curation, Analyzed the data, Writing– Original Draft. Y.C.: Performed experiments, Provided advice and Technical assistance, Writing– Review & Editing, Supervision, Project Administration. Y.H.: Performed experiments. S.X. and N.Z.: Analyzed the data, Provided advice and Technical assistance. H.Z.: Provided advice and Technical assistance. Q.L.: Conceived and Designed the study, Writing–Review & Editing, Project Administration, Funding Acquisition. H.L.: Conceived and Designed the study, Provided advice and Technical assistance, Writing–Review & Editing, Supervision, Project Administration, Funding Acquisition. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Huanying Zheng, Qiliang Liu or Hongbo Liu.

Ethics declarations

Ethics approval and consent to participate

This study was performed in strict accordance with the recommendations in the Guide for the Institutional Animal Care and Use Commission (IACUC). The protocols were approved by the Committee on the Ethics of Animal Experiments of the Guilin Medical University (Number: GLMC202303003).

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Not applicable.

Competing interests

The authors declare no competing interests.

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Qu, Y., Wang, J., Chen, Y. et al. Establishment and application of a wild neonatal mouse model infected with an Echovirus 30 isolate. Virol J 22, 69 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-025-02684-z

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Virology Journal

ISSN: 1743-422X