Coxsackievirus A6 Intragastrically Infected BALB/c Neonatal Murine Model

Background Hand,foot and mouth disease(HFMD)is a common infectious disease caused by various enteroviruses,primarily affecting children under the age of five.HFMD is mainly transmitted through the respiratory tract and presents symptoms such as fever,rash or vesicles on the hands,feet,and mouth.It can also lead to severe complications in the nervous and respiratory systems.The introduction of the enterovirus 71(EV71)vaccine has led to changes in the epidemiological spectrum of HFMD,with Coxsackievirus A6(CA6)emerging as one of the main pathogens.Compared to other HFMD pathogens,CA6 causes atypical symptoms,with a more widespread distribution of skin rashes,severe skin lesions,and the presence of vesicles or even erosive rashes.Nail shedding is also commonly observed.The mechanisms underlying CA6 infection and pathogenesis remain unclear,and there is a lack of targeted drugs and vaccines.The absence of suitable animal models is a significant limitation for studying the mechanisms and developing antiviral drugs and vaccines.The aim of this study is to establish a more representative animal model for CA6 infection.We used 7-day-old BALB/c mice and simulated the development of the disease by intragastrically infecting them with CA6 virus.By observing the clinical symptoms and mortality of the infected animals,detecting virus amplification and pathological changes in various tissues,we confirmed the successful establishment of the model and determined its key biological indicators.Based on this animal model,we further analyzed the host’s immune regulatory mechanisms against viral infection.We analyzed the cell populations that play a dominant role in antiviral defense and the cytokines involved in immune responses.Additionally,we used RNA-Seq technology to identify differentially expressed genes in blood and hind limb muscles at different time points after infection,aiming to explore the molecular mechanisms of immune regulation that influence the CA6 virus infection process.The CA6 mouse infection model established in this study provides a new experimental model and research method for studying the pathogenesis and treatment of the disease.Based on our investigation into the immune mechanisms of CA6 virus infection using this model,our study is expected to offer new insights and targets for the prevention and treatment of related diseases.This will enhance our understanding and improve the management of these diseases.We believe that the results of this research will have a positive impact on the prevention,control,and treatment of HFMD and other related infectious diseases,contributing to the advancement of global public health.Methods We used 7-day-old BALB/c mice and performed intragastrical infection or intraperitoneal injection to infect them with CA6 virus.The intragastrical infection group received 1 × 10^6.5 TCID50 of CA6 virus in a volume of 100 μl,while the intraperitoneal injection group received 1×10^4.5 TCID50 of CA6 virus in a volume of 100 μl.The control groups were administered an equivalent volume of PBS via intragastrical infection or intraperitoneal injection.We observed the clinical symptoms,mortality rate,and changes in body weight of the animals daily after infection.On days 1,3,5,and 7 post-infection,we collected blood,muscle,heart,liver,lung,stomach,small intestine,large intestine,brain,brainstem,and spinal cord samples.Using fluorescence real-time quantitative PCR probe method,we determined the viral load in each tissue to compare the viral amplification in different tissues of the mice.We also conducted H&E staining to examine the pathological changes in the tissues and identify the primary sites of pathological damage.Additionally,we assessed the changes in immune cells and cytokines at different time points to analyze the immune regulatory mechanisms.Furthermore,on days 3,5,and 7 post-infection,we collected blood,muscle,brain,thymus,and spleen tissues for RNA-Seq analysis.This allowed us to identify differentially expressed genes at different time points and screen for pathogenic and immune regulatory genes associated with CA6 infection-induced hand,foot,and mouth disease.Results Mice infected with CA6 virus through intragastrical infection displayed clinical symptoms such as reduced activity and progressive paralysis in the hind limbs.Four days after infection,the mice exhibited a rapid decline in body weight,and by day 8,all mice had died.Viral load measurements in various tissues revealed that the virus could disseminate through the bloodstream to different organs and replicate.The highest viral loads were found in the blood,muscle,and spinal cord.Pathological observations showed that CA6 virus infection caused damage to tissues such as muscle,brain,small intestine,and large intestine,although the extent of damage varied slightly.In the CA6infected mouse model,the virus primarily replicated in the skeletal muscles of the hind limbs,with significantly higher viral loads compared to other tissues.The main histopathological features in skeletal muscles included muscle fiber rupture,necrosis,extensive infiltration of inflammatory cells,and diffuse distribution of viral antigens.By performing flow cytometry analysis to detect the proportion of neutrophils and monocytes in peripheral blood after intragastric infection,it can be observed that the infection leads to an increased number of immune cells in response to CA6 invasion,with the innate immune system playing a dominant role in this process.Immunohistochemistry and immunofluorescence staining indicate the presence of a large number of infiltrating neutrophils and macrophages in muscle tissue during the late stage of infection,causing degeneration and necrosis of muscle cells.Infiltration of neutrophils in the spinal cord suggests that the nervous tissue is also affected by viral infection.Additionally,the overall concentrations of pro-inflammatory cytokines IL-6,IL-18,and IFN-gamma are significantly elevated in the serum,along with the increased number of neutrophils in the blood,indicating a strong inflammatory response in mice following infection.RNA-Seq sequencing analysis of blood,muscle,brain,thymus,and spleen from CA6-infected mice identified differentially expressed genes that were commonly altered at different time points.A total of 1399 differentially expressed genes were found in blood,2674 in muscle,4251 in brain,1676 in thymus,and 1586 in spleen.Functional analysis of these commonly altered genes revealed that in blood and hind limb muscles,genes related to the organism’s response to the virus were upregulated.In blood,differentially expressed genes were also enriched in cytokine-mediated signaling pathways and immune-related pathways such as the response to IL-1β.Particularly in defense against viral and bacterial invasion,the differentially expressed genes were significantly enriched in immune-related biological processes.In addition,DEGs are also involved in molecular functions related to metabolism and immune response,such as ATP metabolism,response to interferon-beta,regulation of innate and adaptive immunity,positive regulation of cytokine production,and more.KEGG analysis revealed that DEGs in blood group are mainly enriched in signaling pathways such as osteoblast differentiation,NOD-like receptor signaling pathway,and JAK-STAT signaling pathway.DEGs in muscle group are primarily enriched in pathways including ECMreceptor interaction,regulation of lipolysis in adipocytes,beta-alanine metabolism,and chemokine signaling pathway.DEGs in spleen tissue are involved in pathways such as phagosome,NF-kappa B signaling pathway,B cell receptor signaling pathway,human cytomegalovirus infection,human T-cell leukemia virus 1 infection,viral myocarditis,human immunodeficiency virus 1 infection,as well as pathways related to natural killer cell-mediated cytotoxicity,antigen processing and presentation,and Ras signaling pathway.Differential expression genes in all five tissues are closely associated with the immune system.Conclusion In this study,a mouse model of coxsackievirus A6(CA6)infection was established using an intragastrical infection method to simulate natural infection routes.The animals exhibited typical symptoms of hand,foot,and mouth disease(HFMD)animal models,including weight loss,hind limb paralysis,and death.Subsequently,virological,immunological,pathological,and transcriptomic analyses were conducted on infected animals,leading to the establishment of the CA6 infection animal model.During the study,the distribution and amplification of the virus in various organs were analyzed,and the tissue pathology was observed.It was found that the virus primarily replicated in skeletal muscles,leading to a large influx of inflammatory cells and necrotizing myositis in the muscles.The nervous tissues and other organs also showed a certain level of viral invasion and lesions.Further analysis using techniques such as Luminex,IHC,and IF revealed a significant increase in inflammatory and proinflammatory cytokines in mice infected with the virus,indicating a severe inflammatory response induced by the viral infection.Transcriptomic analysis was performed on blood,muscle,brain,spleen,and thymus based on the animal model,revealing changes in gene expression related to viral response,metabolism,and immune activation,providing initial insights into the dynamics of gene expression following CA6 infection in mice.The establishment of this mouse model provides a new experimental tool for studying the treatment and prevention of CA6 virus.Through the application of this model,a deeper understanding of the infection mechanisms and pathophysiology of CA6 virus can be achieved,leading to the development of more effective treatment and prevention strategies,and promoting the control and prevention of hand,foot,and mouth disease.