Research progress and application prospects of animal models of group B Coxsackievirus infections

ABSTRACT

Group B Coxsackieviruses (CVBs) consist of six serotypes, CVB1 to CVB6, which can clinically affect the heart, brain, liver, pancreas and other organs, causing myocarditis, encephalitis, myelitis, pancreatitis, hand-foot-and-mouth disease (HFMD) and other diseases, and can even lead to death. CVBs are widespread globally and highly contagious. However, there are currently no approved CVB vaccines or effective treatments. The construction and optimization of animal models will aid in the in-depth understanding of CVB infections and its pathogenesis, providing essential tools for the exploration of vaccine development and antiviral therapies. This paper reviews the latest research progress and application prospects of CVB animal models.

Introduction

Group B Coxsackieviruses (CVBs) belong to the Enterovirus genus within the Picornaviridae family and include six serotypes, CVB1 to CVB6. They are non-enveloped, small (30 nm), single-stranded positive-sense RNA viruses with a genome length of 7.4 kb [1]. CVBs are highly contagious, primarily transmitted through the fecal-oral route and respiratory route, causing infection in patients from all age groups. After infection, CVBs replicate and spread within the gastrointestinal tract, which can lead to systemic infection. Clinically, mild CVB infections can cause fever, hand-foot-and-mouth disease (HFMD) and upper respiratory tract infection (URTI), whereas severe infections can result in viral encephalitis, aseptic meningitis, myelitis, myocarditis, pancreatitis or even death. Notably, CVBs are recognized as a significant pathogen responsible for diseases of the heart, pancreas and central nervous system. Moreover, it has been reported that CVBs may play a role in the development of chronic diseases such as type 1 diabetes mellitus (T1DM)[2–5].

CVBs exhibit widespread endemic characteristics, continuously circulating in regions such as Asia, Europe, Africa and North America. Epidemiological data show that outbreaks of CVB have occurred in several regions of China, including Jiangsu, Zhejiang, Fujian, Hebei, Henan, Shandong, Yunnan and Inner Mongolia[6–13]. The United States also reported prolonged and sustained circulation of CVB from 2009 to 2016[14,15]. In recent years, research on circulating strains of CVB has revealed that the six serotypes exhibit co-circulation and genetic recombination during transmission[16]. Currently, treatments for CVB primarily focus on symptomatic and supportive care, lacking effective therapeutic methods and approved vaccines. Research into the mechanisms of CVB infections and pathogenesis will aid in the development and evaluation of drugs and vaccines. Animal models play a crucial role in this process. This paper reviews the established CVB animal models and their applications in drug and vaccine research.

CVB mouse models

Mouse models have several advantages, including low cost, stable genetic background, simplicity of operation and relatively mature techniques, making them widely used in research. As summarized in Figure 1 and Table 1, in developing CVB mouse models, researchers select different mouse strains, ages and inoculation strategies based on the experimental objectives. Mouse models are primarily divided into infection models and disease models. Infection models involve introducing pathogens into mice to study the infection process, the pathogenic mechanisms of the pathogens, and the host's immune response. On the other hand, disease models introduce pathogens to induce specific diseases in mice to study the pathogenesis, disease progression, and treatment methods of those diseases. Commonly used mouse strains include BALB/c, C57BL/6 and NOD. When choosing the age of the mice, they are primarily categorized into neonatal and adult mice. Due to their susceptibility, neonatal mice aged 1–3 days are often used to model CVB infections, while adult mice are typically used to model CVB diseases.

Figure 1.

Animal models used in the study of major human diseases caused by CVB. CVB attacks multiple human organs causing severe diseases. The figure displays the main animal models for different serotypes of CVB-induced diseases.

Table 1.

CVB mouse models.

Strain	Gender	Age	CVB isolate	Inoculation route	Inoculation titer	Clinical symptoms and disease	Ref.
BALB/c	N/A	1 day	CVB1-204	i.p.	1.00E-03 (T)	inactivity, emaciation, limb weakness, hair thinning, hunching and death	[17]
CD-1	N/A	2 days	CVB1 (V1-001)	i.p.	1.00E + 02 (P)	acute infectious myositis occurred in both nu/nu and nu/+ mice, but only nu/+ mice developed chronic weakness and myositis	[25]
ddY	N/A	1 day	CVB2-Ohio-1	i.c.	N/A	dome-shaped heads, absence of the cerebral cortex, apoptosis and necrosis in the cortex	[22]
BALB/c	N/A	3 days	CVB2-YN31V3	i.c.	1.00E + 5.5 (T)	hind limb paralysis, lost weight and pathological changes in the brain, kidneys, hind limb muscles, heart, liver, and pancreas	[18]
BALB/c	Male	4 weeks	CVB2-04/243 CVB2-04/279 CVB2-Ohio-1	i.p.	2.50E + 04 (P)	myocarditis developed and extensive pancreatic inflammation was observed in all mice infected with CVB2/04/279 or CVB/O	[39]
BALB/c	N/A	1 day	CVB3	i.c.	2.00E + 05 (P)	central nervous system diseases, death	[19]
ICR	N/A	14 days	CVB3-Nancy	i.p.	2.60E + 04 (T)	dilated cardiomyopathy	[23]
C57BL/6	Male	pregnant	CVB3-Nancy	i.p.	1.00E + 06 (T) 2.50E + 06 (T) 5.00E + 06 (T)	ongenital heart defects, ventricular septal defect, abnormal myocardial architecture, double-outlet right ventricle	[29]
BALB/c	Male	3–4 weeks	CVB3m	i.p.	1.00E-03 (T) 1.00E-02 (T) 1.00E-02 (T)	death rate and the heart weight increased, cardiac function decreased	[32]
C57BL/6	Male	5–6 weeks	CVB3-Nancy	i.p.	1.00E + 05 (T)	myocardium inflammatory infiltration, body weight decline and death	[40]
C57BL/6	Male	8 weeks	CVB3-Nancy	i.p.	3.50E + 01(P)	PKO mice only exhibited mild myocarditis, while perforin-positive mice showed extensive myocardial damage and myocardial fibrosis	[41]
C57BL/6	Male	10–15 weeks	CVB3-Nancy	p.o.	5.00E + 07 (P)	testosterone enhances CVB3 lethality	[43]
NOD	Female	4 weeks 8 weeks	CVB3-M, CVB3-20,CVB3-ZU, CVB3-AS,CVB3-O, CVB3-CO,CVB3-GA,CVB3-Edwards, CVB4-JVB	i.p.	5.00E + 05 (T)	N/A	[46,47]
A/J	Male	5 weeks	CVB3-Nancy	i.p.	2.00E + 05 (P)	cardiac mucsle injury	[49]
C3H	Male	3 weeks	CVB3-Nancy	i.p.	1.00E + 05 (P)	cardiomyocyte damage, inflammation, fibrosis and arrhythmia	[50]
C3H(SCID)	Male	18-21 days	CVB3/20,CVB3/0	i.p.	1.00E + 05(T)	Both viruses caused myocarditis	[51]
ABY/SnJ	N/A	N/A	CVB3-Nancy	N/A	N/A	N/A	[52]
SWR; Balb/c	Male	5 weeks	CVB3-Nancy	i.p.	1.00E + 02-1.00E + 07 (P)	SWR mice were more sensitive to CVB3-induced myocardit	[53]
DBA/2	Male	5 weeks	CVB3-H3	i.p.	2.00E + 03(P)	chronic viral myocarditis	[54]
ICR	N/A	2 days	CVB4 (Dowell)	i.p.	1.00E-04 (T)	residual paralysis, thinning of myocardial wall, ventricular aneurysms, myofiber necrosis and/or fibrous replacement, white epicardial scar	[24]
ICR	N/A	3 days	CVB4-LY114F	i.m.	1.00E-07 (T)	cerebral cortical neuron edema, myocardial lymphocytic infiltration	[26]
Swiss	Female	pregnant	CVB4-E2	i.p.	2.00E + 05-2.00E + 06 (T)	N/A	[27,28]
BALB/c	Male	4 weeks	CVB4-V	i.p.	1.00E + 02 (P)	severe pancreatitis and chronic pancreatitis	[37,38]
NOD	Female	8 weeks	CVB4-Edwards	i.p.	5.00E + 05 (P)	CVB4 RNA persists in the kidneys for at least 14 days, and the absence of TLR3 increases the abundance of CVB4 in the kidneys	[48]
BALB/c	N/A	3 days	CVB5-JS417	i.p.	3.16E + 07 (C)	necrosis of neuronal cells	[20]
BALB/c	N/A	1 day	CVB6-XZ-1999, CVB6-XZ-2000	i.p.	1.00E + 04-1.00E + 06 (T)	weight loss, limb paralysis and death	[21]

i.p., intraperitoneal; p.o., peros; i.c., intracranial; i.v., intravenous injection; i.m., intramuscular; T, TCID₅₀; C,CCID₅₀; P, Pfu; N/A, not applicable.

Neonatal mice

Neonatal mice are frequently used to construct animal infection models related to CVBs. Among these, inbred strains such as BALB/c and ddY, and outbred strains like Swiss, ICR and CD-1, are the most common neonatal mouse strains used in experiments. Neonatal mouse models based on infections from all six serotypes of CVBs have been studied and reported. In recent studies, BALB/c neonatal mice are often chosen due to their susceptibility to CVB and stable characteristics. Yin et al., in order to better evaluate the efficacy of antiviral agents and vaccines against CVB1, infected 1-day-old BALB/c neonatal mice with the CVB1-204 strain via intraperitoneal injection, inducing clinical symptoms in neonatal mice such as reduced activity, emaciation, limb weakness, sparse fur, and a hunched back, with a mortality rate reaching 100%, providing an effective CVB1 neonatal mouse infection model[17]. A study analyzed the CVB2-YN31V3 strain isolated in 2019 from a HFMD patient in Yunnan. It was found that this strain has a 12.91% nucleotide sequence difference from its closest strain, indicating it may be a recombinant virus. It can induce significant disease symptoms in 3-day-old BALB/c mouse models, and histopathological analysis revealed severe pathological changes in multiple tissues of the virus-infected mice[18]. Ralph et al. conducted a study on the susceptibility of the central nervous system in newborns to CVB infections. They constructed a recombinant virus that expresses enhanced green fluorescent protein (eGFP-CVB3) and performed intracranial injections on 1-day-old BALB/c mice. The results showed that the recombinant virus heavily targeted cells with stem cell characteristics in the brains of the newborn mice. As these cells differentiated, the virus progressively invaded neurons and the brain parenchyma, leading to the development of central nervous system diseases. Additionally, the mortality caused by viral infection was age-dependent, with 1-day-old mice exhibiting a 100% mortality rate, 3-day-old mice a 50% mortality rate, and 4-day-old mice a 20% mortality rate. One-week-old mice were able to survive without fatal injuries[19]. Mao et al. used a CVB5 clinical isolate (CVB5/JS417) to infect 3-day-old BALB/c mice, establishing the first neonatal mouse model of CVB5. The model revealed that mice infected with CVB5/JS417 exhibited neuronal cell necrosis in multiple tissue sites. Monitoring viral loads at different time points showed that CVB5 initially replicated in the small intestine and then disseminated to various organs through viremia. Consequently, the virus may potentially enter the brain via spinal cord axons[20]. Recently, a new study successfully established a CVB6 mouse infection model that induced clinical features such as weight loss, limb paralysis, and death in BALB/c neonatal mice[21]. ddY mice are another inbred strain characterized by a healthy immune system and genetic stability, commonly used in various research studies. In 2019, Ushioda et al. utilized the CVB2 prototype strain (CVB2-Ohio-1) to develop an animal model infecting ddY neonatal mice. Histopathological examination showed that the infected mice exhibited brain damage, with magnetic resonance imaging (MRI) revealing cortical atrophy two weeks post-infection. CVB2 replicated in mature neurons in the cortex, hippocampus, thalamus and midbrain, causing activation of microglia and astrocytes, leading to cortical cell apoptosis, severe necrosis and lateral ventricle enlargement[22].

In addition to inbred mice, researchers use outbred mice as subjects for CVB neonatal mouse models. As early as the 1980s, researchers used CD-1 and ICR mice to construct infection models for CVB1, CVB3 and CVB4, indicating that CVBs may be associated with the development of necrotizing myocarditis and dilated cardiomyopathy [23,24]. Additionally, under immune system mediation, there can be a transition from acute viral infection to systemic chronic myositis[25]. A study successfully established a CVB4 animal infection model by intramuscular injection in 3-day-old ICR mice, inducing acute myocarditis and cerebral cortical neuron edema[26]. The construction of this model could potentially aid in the evaluation of candidate vaccines and potential antiviral drugs against CVB4 infection.

Additionally, researchers explore congenital viral infections in neonatal mice by infecting pregnant mice during gestation. In a study, Hela et al. used outbred Swiss mice to investigate the vertical transmission of CVB4 between mother and fetus. This model showed that CVB4 can be transmitted prenatally via the placenta and perinatally during delivery, with the virus potentially persisting in neonatal mice[27]. Further research showed that neonatal mice infected with CVB4 in utero exhibited reduced expression of self-antigens, increasing the risk of developing autoimmune diseases[28]. A study using a C57BL/6 pregnant mouse model also demonstrated that CVB3 can inhibit the proliferation of cardiomyocytes by stimulating the TGF-β1 pathway, leading to congenital heart defects in mouse fetuses[29]. These findings highlight that the threat of CVB to newborns extends beyond the neonatal period and may induce congenital diseases during the fetal stage.

In summary, CVB neonatal mouse infection models have garnered extensive attention and research. These models help simulate viral attacks on humans and provide crucial tools for the development and evaluation of therapeutic drugs and vaccines. However, due to physiological and developmental differences between neonatal and adult mice, it can be challenging to directly extrapolate findings from neonatal mice to adult mice or humans. Additionally, due to the smaller size and increased fragility of neonatal mice compared to adult mice, extra care must be taken during experimental procedures to minimize data variability caused by handling and inconsistent growth and development of the mice.

Adult mice

Although adult mice have lower susceptibility to CVB, different strains of mice exhibit varying degrees of susceptibility, making them valuable in studying CVB-related diseases.

BALB/c

BALB/c mice are a common strain in CVB mouse infection and disease models. Their genetic background is inbred, leading to minimal individual variation and stable genetic traits, and they are moderately susceptible to CVB. Beyond neonatal mice, adult BALB/c mice aged 3–6 weeks are widely used in constructing myocarditis and pancreatitis disease models.

Myocarditis is a cardiovascular disease, with viral myocarditis being the most common type caused by viral infections. Clinically, viral myocarditis can lead to severe symptoms such as arrhythmias and sudden death, and it can also progress to chronic heart failure. CVBs have been confirmed to be closely associated with viral myocarditis[30]. In the 1990s, Kandolf et al. constructed a model of persistent CVB3 infection in athymic mice. They confirmed the presence of CVB3 RNA in the myocardium through in situ hybridization, and found that the myocardium of infected mice was affected by the virus in a diffuse and multifocal manner, thereby confirming that CVB3 is a serotype capable of causing viral myocarditis[31]. Nie et al. established a viral myocarditis animal model by repeatedly infecting BALB/c mice with increasing doses of a variant of CVB (CVB_3m). Compared to a single infection model, mice subjected to repeated infections exhibited significant ventricular remodelling characteristic of cardiomyopathy[32]. Researchers also have compared the immune responses to CVB3 infection in C57BL/6 and BALB/c mice, finding that the virus clearance rate in the heart tissue of BALB/c mice is slower than in C57BL/6 mice[33]. Detection of virus-specific antibody responses revealed that IgG levels in C57BL/6 mice were significantly higher compared to BALB/c mice[33]. The results suggest that virus-specific IgG may play an important role in CVB3 clearance in C57BL/6 mice.

Pancreatitis is a digestive system disease clinically characterized by inflammation, edema and necrosis of the exocrine pancreas. As early as the 1950s, researchers identified a link between CVB and the occurrence of pancreatitis[34,35]. Further research showed that in a survey of 118 patients with acute and recurrent chronic pancreatitis, 34% (40 cases) of the patients exhibited elevated CVB antibody titres, primarily targeting CVB3 and CVB4[36]. Ramsingh et al. employed a virulent variant of CVB4 (CVB4-V) to establish a chronic pancreatitis model in BALB/c mice and investigate the regulatory role of interleukin-12 (IL-12) on CVB4-V infection. Their results indicated that IL-12 can prevent virus-induced tissue damage and the development of chronic pancreatitis[37]. Subsequently, they explored the role of interleukin-10 (IL-10) using this animal model and found that IL-10 contributes to the progression of chronic inflammatory disease in CVB4-V infected BALB/c mice[38]. Furthermore, a study tested two clinical isolates from Korea (CVB2-04/243, CVB4-04/279) and the CVB2-Ohio-1 (CVB2-O) strain on a 4-week-old BALB/c mouse model. Histopathological examination results showed that all mice infected with either CVB4-04/279 or CVB2-O developed myocarditis and extensive pancreatic inflammation, while mice infected with CVB2-04/243 did not exhibit these symptoms. This provides a useful CVB2 adult BALB/c model for reference[39].

The BALB/c mouse model in CVB research, due to its genetic homogeneity, heightened sensitivity and substantial immunological data, ensures stable experimental results and facilitates in-depth studies of immune responses. However, species differences, linited genetic diversity, and variations in organ structure restrict the extrapolation of research findings to human applications.

C57BL/6

C57BL/6 mice, produced through inbreeding, are black in colour and are known for their stable strain characteristics and ease of breeding. Although C57BL/6 mice have lower susceptibility to CVB, their strong immune system response and suitability for gene knockout and transgenic strain development make them commonly used in the construction of mouse models, particularly for the study of multigenic interactions and complex disease models. Li et al. developed a C57BL/6 infection model using the CVB3-Nancy strain and found that initial body weight and virus infection dose significantly influence the incidence of viral myocarditis in these mice. The low body weight group (Group A, 11–15 g) exhibited a higher mortality rate (40%–100%), whereas the high body weight group (Group B, 18–20 g) maintained a relatively stable survival rate. Additionally, myocardial inflammatory infiltration worsened with increasing virus dose. When the initial body weight was 18–20 g and the virus inoculation dose was 30,000 TCID₅₀, the mice developed severe myocardial inflammation with disorganized myocardial fibre arrangement[40]. A study investigating the relationship between perforin and CVB3 utilized gene knockout mice. Researchers targeted the perforin gene in C57BL/6 mice, resulting in perforin knockout (PKO) mice. In the study, PKO mice developed only mild myocarditis and resolved the condition quickly after viral infection. In contrast, perforin-positive mice exhibited extensive myocardial lesions and progressed to severe myocardial fibrosis in the late stages of the disease. This indicates that perforin is an important factor in the development of viral myocarditis[41]. Interferon receptor gene knockout (Ifnar-/-) mice baesd on the C57BL/6 strain are immunodeficient models sensitive to a variety of viral studies. These models are used to investigate the pathogenic mechanisms of viruses and to develop antiviral vaccines and drugs[42]. A recent study aimed to determine whether testosterone could promote CVB3 replication by orally infecting male and female C57BL/6 Ifnar-/- mice. The results showed that mice in the testosterone-treated group shed significantly more CVB3 in their feces compared to the placebo-treated group. Testosterone-treated male mice had a 100% mortality rate after viral infection, whereas testosterone-depleted male mice showed a significantly reduced mortality rate of about 50%. This indicates that testosterone enhances enterovirus replication. Further analysis revealed that testosterone can affect the immune response to the virus[43]. This study provides a potential explanation for the observed sex differences in the epidemiology of CVB infections.

NOD

NOD mice are commonly used animal models for diabetes research, possessing immune system dysregulation characteristics similar to those in humans and being sensitive to CVB. Therefore, NOD mice are suitable for studying how CVB triggers or influences autoimmune responses. The NOD mouse model was first introduced by Makino et al. in 1980. This strain of mice exhibits spontaneous diabetes with a gender disparity before reaching 30 weeks of age, with an incidence rate of 80% in females and 20% in males[44]. Studies have reported that CVB4 accelerates the onset of T1DM in the NOD mouse model, leading to early onset in nearly half of the mice. This may be related to dysbiosis caused by enterovirus infection[45].

Currently, most enterovirus studies based on the NOD mouse model mainly use mice aged 8–10 weeks. However, one study found that using NOD mice of different ages has varying effects on the incidence of T1DM. The findings revealed that inoculating the mice at younger ages (around 4 weeks) significantly reduced the occurrence of T1DM, with the incidence rate dropping by 2–10 times. However, when older NOD mice (around 8 weeks) were inoculated with the virus, they rapidly developed T1DM. This phenomenon was consistent across all nine CVB strain experimental groups[46]. Researchers further explored this phenomenon and suggested that CVBs do not induce T1DM in young mice, possibly due to the production of interferons by insulin-producing cells or the lack of Coxsackievirus and Adenovirus Receptor (CAR) expression. Additionally, a chimeric CVB3 virus carrying the IL-4 gene, while capable of replicating within the islets, significantly reduced the likelihood of causing T1DM[47]. Walter et al. evaluated renal damage in NOD (Tlr3^+/+, Tlr3^-/-) mice following intraperitoneal injection of CVB4. The results indicated that CVB4 RNA was still present in the glomeruli 14 days after inoculation, and the absence of TLR3 increases the abundance of CVB4 in the kidney. Additionally, CVB4 infection led to initial changes in renal gene expression, predisposing the mice to the development of T1DM. This phenomenon may be closely related to the activation of the TLR3 pattern recognition receptor[48].

Studies using the NOD mouse model have shown that the relationship between CVB and T1DM is complex, influenced by factors such as the timing of infection and the characteristics of the viral strain. These studies provide important insights into the mechanisms by which viruses can induce autoimmune diseases, and may offer a scientific basis for future strategies in the prevention and treatment of T1DM. Future research will need more detailed mechanistic investigations and larger-scale clinical trials to clarify the exact role of CVB in the onset of T1DM.

Other adult mice

In addition to the commonly used mouse models mentioned above, some adult mice have been employed in research on CVB, particularly CVB3. For example, A/J, C3H, C3H SCID, and ABY/SnJ mice have been used in the establishment of myocarditis models and mechanism studies related to CVB3[49–52]. Although BALB/c mice are commonly used in current research, more suitable model animals may still be awaiting discovery in special circumstances. Researchers have compared myocarditis induced by the CVB3-Nancy strain in SWR mice with the traditional BALB/c mouse model, finding that SWR mice inoculated with CVB3 developed more severe myocarditis but milder pancreatitis compared to BALB/c mice. Notably, the body weight of SWR mice was not affected by CVB3 inoculation, suggesting that the SWR mouse model might be ideal for studies involving high doses of CVB3[53]. Furthermore, in research on dilated cardiomyopathy induced by CVB3, due to BALB/c mice's tendency to develop acute myocarditis, researchers have chosen DBA/2 mice as a more suitable model[54]. In conclusion, we still need to continue exploring the possibilities of CVB in animal models to construct more specific and precise models.

CVB non-mouse models

Although mouse models offer advantages in terms of acquisition and handling, significant heterogeneity exists between mice and humans in disease development, making it impossible to accurately replicate all clinical manifestations. As research advances, the establishment of infection models is continually being optimized to more closely resemble the process of viral infection in humans. To explore CVB animal infection models that are closer to humans, researchers have attempted to construct models in rats and non-human primates using natural infection routes. As summarized in Figure 1 and Table 2, these models currently include Syrian golden hamsters, baboons, crab-eating macaques, squirrel monkeys and rhesus macaques.

Table 2.

CVB non-mouse models.

Strain	Gender	Age	CVB isolate	Inoculation route	Inoculation titer	Clinical symptoms and disease	Ref.
Syrian golden hamsters	Male	3 weeks	CVB1	nasal drip	1.00E + 7.25 (C)	listlessness, decreased body temperature, red rash and herpes	[55]
Syrian hamster	Female	4 weeks	CVB3 (A278741)	i.p., nasal drip	1.00E + 5.57 (C)	pharyngitis, cutaneousherpes, reduced appetite, restricted activity, increased body temperature, decreased body weight	[56]
Rhesus monkey	Female	3 months	CVB3 (A278741)	nasal drip	2.00E + 6.75 (C)	decreased activity, lowered body temperature, reduced body weight, HFMD lesions characteristic on the skin mucosa, cutaneous herpes	[56]
Papio papio	Female	N/A	CVB3m	i.p.	4.00E + 08 (P)	delayed hypersensitivity and myocarditis	[57]
Macaca fascicularis	Female	7.3–9.5 years	CVB3-H3, CVB3-MCH	i.v.	1.00E + 06–1.00E + 07 (P)	meningitis, encephalitis, pancreatitis, hepatitis	[59]
Squirrel monkey	N/A	6–229 days	CVB4	i.v., i.p.	1.00E + 6.5 (T)	mitral valve lesions, aortic valve	[58]

i.p., intraperitoneal; i.v., intravenous injection; T, TCID₅₀; C,CCID₅₀; P, Pfu; N/A, not applicable.

Syrian golden hamsters

Syrian golden hamsters exhibit disease symptoms, pathogenesis and immune responses that closely resemble those of humans, making them widely used in studies related to viral infections.

In 2023, Li et al. aimed to better simulate the natural infection route of the virus by using intranasal instillation to construct a CVB1 infection model in Syrian golden hamsters[55]. The model showed that within 14 days of infection, the hamsters exhibited symptoms to varying degrees such as lethargy, reduced body temperature and herpetic lesions, resembling human HFMD. This model provides a valuable reference for the study of HFMD caused by CVBs. In another study on HFMD caused by CVB3, Duan et al. also used Syrian golden hamsters as experimental animals to explore and optimize CVB infection models[56]. They found that hamsters infected with the virus via intranasal instillation developed skin herpetic lesions, reduced appetite, limited activity, elevated body temperature and weight loss, which are consistent with human HFMD symptoms. Furthermore, when comparing intraperitoneal injection and intranasal instillation as infection methods, they discovered that while both methods induced severe symptoms in hamsters, intranasal instillation produced more severe symptoms[56]. The Syrian golden hamster model has significant advantages in CVB infection modelling. It can simulate the mild symptoms and clinical manifestations of HFMD caused by CVBs in humans, which mouse models currently cannot achieve. Further refinement and development of the Syrian golden hamster model will help advance the research into the mechanisms of mild diseases related to CVBs.

Non-human primates

Non-human primates are the most phylogenetically similar to humans and share many similarities in their immune and physiological systems. As a result, data from non-human primate models are more convincing in simulating human clinical diseases and their treatments. Currently, non-human primate models for CVB have been constructed, using species such as baboons, crab-eating macaques, squirrel monkeys and rhesus macaques.

As early as the twentieth century, researchers began conducting CVB-related experiments in non-human primates. In 1981, Ronald et al. systematically evaluated the cell-mediated immunity of CVB3-induced myocarditis in baboons. They pointed out that similar phenomena are likely to occur in humans[57]. Subsequently, a study inoculated squirrel monkeys with CVB4 via intravenous or intraperitoneal injections. Out of nine monkeys, three developed mitral valve lesions, and one of these monkeys also exhibited aortic valve damage, indicating that CVB4 can cause valvular lesions in non-human primates[58]. This suggests that CVB4 may cause similar pathogenicity in humans.

Crab-eating macaques offer the advantages of a short breeding period, ease of cultivation and physiological similarities to humans. In 2013, Cammock et al. inoculated crab-eating macaques with CVB3 to construct a related animal model. Virological studies conducted 28 days post-inoculation revealed that all macaques produced neutralizing antibodies, and CVB3 was detected in their plasma. Additionally, the experimental group that received a high dose of the virus exhibited severe disseminated disease, whereas animals inoculated with a low dose or via oral infection showed no significant symptoms. Necropsy results indicated that all crab-eating macaques exhibited signs of myocardial inflammation and damage, highlighting the prevalence of myocardial injury in enterovirus infections[59].

In a study on HFMD caused by CVB3, Duan et al. constructed a CVB3 animal infection model using both Syrian golden hamsters and rhesus monkeys. In this model, rhesus monkeys infected with CVB3 via intranasal instillation exhibited clinical symptoms similar to human HFMD, including mild pathological lesions. The disease progression and virus clearance in these monkeys were largely consistent with those observed in humans. Duan et al. highlighted that non-human primates exhibit physiological and anatomical similarities to humans, particularly in terms of susceptibility to pathogens, making them advantageous for establishing animal models to study viral infections[56].

Using non-human primate models can help address the limitations and heterogeneity of mouse models, providing us with more comprehensive information about the pathogenic mechanisms of CVBs. This approach offers a valuable platform for the development of related drugs and vaccines, and it is worth further exploration.

Applications of CVB animal models

Currently, there are no effective antiviral drugs or vaccines against CVBs, making the development of such treatments and vaccines crucial for the prevention and control of CVB infections[60]. As summarized in Figure 1 and Table 3, CVB animal models play a crucial role in evaluating the mechanisms of action, therapeutic efficacy, and toxic side effects of drugs and vaccines. They are essential for ensuring that research products can safely and effectively used in humans.

Table 3.

Animal models of CVB for drug and vaccine research.

Strain	Gender	Age	CVB isolate	Inoculation route	Application	Ref.
BALB/c	Male	N/A	CVB3m	N/A	Low-dose administration of plecon-aril continuously can protect mice from fatal harm	[61]
BALB/c	Male	3 weeks	CVB3	i.p.	Astragalus has protective effects on SERCA2 and the endothelin system in mice	[63,64]
CD-1	Male	3 weeks	CVB4-E2	i.p.	Continuous injection of fluoxetine (10 mg/kg/day) can reduce the levels of viral particles in the hearts and pancreasses of mice infected with CVB4	[66]
A/J	Female	6–8 weeks	CVB1, CVB3, CVB4-E2	i.p.	The Mt10 vaccine provides complete protection against myocarditis and pancreatitis induced by the Wt-CVB3 strain, also offers cross-protection against the CVB1 and CVB4	[67,68]
Swiss-albino	Female	5 weeks	CVB4-E2, CVB4-JBV	i.p.	The VLP vaccine designed based on CVB4 VP1 can protect mice from CVB4 infection	[69]
C57BL/6J, NOD, BALB/c, SOCS-1-tg, Rhesus macaques	N/A	N/A	CVB1-6	i.p.	The vaccine has good safety in both mouse models and non-human primates, and is capable of inducing strong neutralizing antibody responses against six serotypes without adjuvants	[70]

i.p., intraperitoneal; N/A, not applicable.

Application of CVB animal models in drug research

Pleconaril is currently the most extensively studied antiviral compound against picornaviruses[61,62]. In 1999, Daniel et al. induced an 80% mortality rate in adult BALB/c mice by intraperitoneally injecting them with CVB_3m. Using this model, they treated the mice with different doses of pleconaril and found that a low dose (20 mg/kg/day) protected the mice from fatal disease[61]. The drug significantly reduced viral levels in the target tissues of infected animals and displayed a dose-dependent effect. Subsequent studies also demonstrated the efficacy of pleconaril, however, due to concerns about its safety and side effects, more comprehensive drug evaluations are still necessary.

In recent years, researchers have shifted their focus to traditional Chinese medicine, which tends to have milder therapeutic effects. Astragalus, a traditional Chinese herb, is widely used in the treatment of cardiovascular diseases. In 2006, Chen et al. constructed a myocarditis mouse model by intraperitoneally injecting 3-week-old male BALB/c mice with 2 × 10⁴ TCID₅₀/0.1 mL of CVB3. They verified the therapeutic effects of Astragalus in this model and found that it had protective effects on the activity of sarcoplasmic reticulum calcium ATPase (SERCA2) and the function of the endothelin system in the mice[63]. Additionally, the effects of Astragalus were found to be similar to those of perindopril, an ACE inhibitor[64]. The concept of drug repurposing has also garnered widespread attention in recent years. Drug repurposing involves using existing drugs to treat new diseases. In 2012, Zuo et al. screened a molecular library and identified fluoxetine (a selective serotonin reuptake inhibitor), demonstrating its potent antiviral activity against various CVB serotypes (B1, B2, and B3) in vitro. Fluoxetine showed potential in inhibiting viral replication, making it worthy of investigation as a potential antiviral drug[65]. Building on this foundation, Benkahla et al. constructed a mouse infection model by infecting CD-1 mice with CVB4-E2 and validated the in vivo therapeutic effects of fluoxetine in this model. The results showed that intraperitoneal injection of fluoxetine (10 mg/kg/day) significantly reduced viral particle levels in the hearts and pancreases of CVB4-infected mice[66]. Although numerous studies have proposed drugs that may combat CVB infections, issues concerning the effectiveness, safety, and usage standards of these drugs still require further validation.

Application of CVB animal models in vaccine research

In recent years, several research teams have been working to develop monovalent or multivalent vaccines against CVBs. In 2021, Reddy et al. utilized an attenuated strain, Mt10-CVB3, to design and develop the Mt10 vaccine. They found that this vaccine could prevent myocarditis and pancreatitis caused by CVB3 and CVB4 in virus-susceptible A/J mouse models. Additionally, in NOD mice, the vaccine provided protection against CVB4-induced T1D[67,68]. This demonstrated the preventive potential of the vaccine and promoted the development of monovalent CVB vaccines. An evaluation study of a VLPs (virus-like particles) vaccine using the Swiss-albino mouse model. Researchers developed a VLP vaccine based on the VP1 region of the CVB4-E2 strain and assessed its protective efficacy in mice. The results showed that the VLP vaccine elicited a robust immune response and provided protection against both the wild-type CVB4-JBV and the diabetes-inducing CVB4-E2 strains, thereby protecting the mice from fatal challenges[69].

Additionally, Stone et al. conducted a study on a multivalent CVB vaccine, utilizing three mouse strains (C57BL/6J, NOD and BALB/c) and rhesus macaques to develop animal models for testing. The results showed that vaccination in both mouse models and rhesus macaques did not result in weight loss or other clinical symptoms. Moreover, it induced strong neutralizing responses against all six CVB serotypes in most animals, indicating good safety and immunogenicity. In the NOD mouse model, the vaccine exhibited preventive effects against CVB infections and protected against pancreatitis. In the BALB/c mouse myocarditis model, the vaccine protected mice from acute CVB3 infection in the heart. Furthermore, in the SOCS-1-tg mouse model–based on NOD mice (where SOCS-1-tg mice express the cytokine signal transduction inhibitor Socs1 under the control of the insulin promoter, making β-cells incapable of producing antiviral IFN responses and thus more susceptible to CVB infection and virus-induced disease) the vaccine also showed protective effects against CVB-induced diabetes[70]. Currently, the research team has published a new multivalent vaccine (PRV-101) targeting five key CVB strains associated with T1DM autoimmunity. This vaccine has achieved positive results in Phase I clinical trials and is expected to become the first multivalent vaccine to prevent CVB infections[71].

Discussion and future perspectives

Currently, the threat of CVBs to human health remains an urgent issue to be addressed. CVB can replicate in multiple organs within the body, causing a variety of clinical diseases and posing significant risks to humans, particularly children and infants, who are especially vulnerable. Researches indicate that, in addition to causing acute clinical diseases, persistent CVB infections carry the risk of leading to chronic diseases. Although extensive researches have been conducted on CVB therapeutic drugs and vaccines, the lack of precise understanding of the viral pathogenic mechanisms has hindered the development of effective treatments. In the process of drug and vaccine research, in vitro experiments can only partially reflect their efficacy. To more comprehensively evaluate the effectiveness of drugs or vaccines, in vivo validation and clinical trials are necessary to ensure their safety and efficacy.

Animal models are essential tools for studying pathogenesis, mechanisms of disease onset, protective mechanisms, drug screening and vaccine efficacy, particularly important in the development of drugs and vaccines. Establishing an animal model that can stably present human clinical diseases or symptoms, and even lead to death, can significantly promote the development and application of drugs and vaccines. Mouse models are currently the most widely used animal models, providing a controlled experimental environment that allows precise adjustment of research variables such as dosage, infection routes, and time points. Additionally, compared to other advanced models, mice have the advantages of low maintenance costs, fast breeding rates, and suitability for large-scale laboratory use. Mice are also easy to genetically modify, for example, through transgenic or gene knockout technologies, enabling researchers to explore the impact of specific genes on viral infection and disease progression. For CVBs, laboratory mice such as BALB/c and C57BL/6 are susceptible. After infection, they may develop myocardial, pancreatic damage, along with other systemic symptoms, which are similar to the clinical manifestations observed in humans. Immunodeficient mice can also be used to increase the incidence of diseases after CVB infections, making them classic models for studying specific diseases. For example, NOD mice, as a spontaneous model of autoimmune diabetes, play an irreplaceable role in studying the mechanism of CVBs-induced T1D and in drug and vaccine evaluation.

Notably, neonatal mice are more sensitive to CVB, and infected neonatal BALB/c mice may experience acute mortality, allowing researchers to observe the progression and outcomes of the disease in a shorter time frame, thereby improving experimental efficiency. However, their short lifecycle also limits the study of the long-term effects and chronic disease progression of CVB infection. Additionally, neonatal mice may exhibit significant individual differences in health status and immune background after birth, which can affect the reproducibility and consistency of experimental results.

However, mouse models have certain limitations, primarily in the following areas: 1. Differences in physiology and immune responses: There are significant differences in physiological structure and immune responses between mice and humans, making it difficult to fully extrapolate research results to humans. 2. Limitations of immunodeficient models: Although immunodeficient mice can be used to increase the incidence of diseases following CVB infection, these models may not accurately reflect the response of a normal immune system to the virus, thus limiting the study of disease mechanisms under normal conditions. 3. Insufficient pathological complexity: Mouse models may not fully replicate the complex pathological changes seen in human infections. In comparison, non-human primate models more closely simulate the characteristics of human viral infections, but their high maintenance costs and ethical issues limit their widespread use.

In summary, addressing the current limitations of animal models is essential. In the future, diversified and multi-level research can be conducted, including quality-controlled standardized models, natural infection route models, co-infection models, and multi-gene models. Additionally, we can further explore the potential of developing non-mammalian models (such as zebrafish, C. elegans, Drosophila, etc.). These models have unique advantages in high-throughput screening, rapid experiments, and low maintenance costs, and they may provide new approaches in large-scale high-throughput drug screening, virus infection dynamics, and host immune responses. By combining the use of various animal models, researchers can gain a more comprehensive understanding of CVB infection mechanisms, providing valuable scientific data for the development of new antiviral drugs and vaccines.

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 82272310 and 82472253), the China Postdoctoral Science Foundation (No. 2022M712666 and 2022T150550), the Beijing Natural Science Foundation (No. L234006), and the the Fundamental Research Funds for the Central Universities (No. 20720220006).

Disclosure statement

No potential conflict of interest was reported by the author(s).