• Some enteroviruses cause central nervous system disease with clinical and pathological features. • Cellular receptors and host immune responses are required for enterovirus entry into the central nervous system. • The pathways and mechanisms by which viruses invade the central nervous system need to be better understood.

• Scientific question Echovirus type 30 (E30) is a human enterovirus species B (EV-B) serotype that can cause severe central nervous system diseases such as encephalitis, aseptic meningitis (AM), and acute flaccid paralysis (AFP). Novel E30 variants have invariably undergone recombination with other EV-B types before their emergence. Studying the recombination model of novel E30 variants is crucial. • Evidence before this study Before 2017, E30 was rare in Ningxia Hui Autonomous Region. In 2017, we observed a sudden increase in E30: five strains were isolated from the AFP case surveillance system, and four strains were isolated during an AM outbreak in October. To understand the cause of the sudden increase, the whole genome sequence of the virus was performed to understand its gene characteristics and its relationship to the disease. • New findings In this study, we found that the E30 strains circulating in Ningxia belong to two lineages (H and J). This study confirmed that the E30 strains in Ningxia in 2010, 2012, and 2016 had different recombination patterns and were recombined with different enteroviruses. • Significance of the study The 2017 epidemic E30 originated from another new lineage with a complex recombination pattern and formed an independent transmission chain in Ningxia.

• Significance of the study: This study indicated that the CVA6 VLP vaccine has good immunoprotective effects against both the D3a and D3b evolutionary branches of CVA6, providing corresponding data support for the development of CVA6 vaccine in China. • Scientific question: D3 genotype coxsackievirus A6 (CVA6) is the main pathogen leading to the outbreak of hand, foot and mouth disease in children in China, causing a serious disease burden. At present, there is no CVA6 vaccine available for epidemic prevention and control. • Evidence before this study: Virus-like particle (VLP) vaccine is a new type of vaccine that does not contain viral nucleic acid, has high safety, and is non-infectious. In the studies of enterovirus A71 (EV-A71), CVA16, CVA10, and other enterovirus VLP vaccines, it has been confirmed that they have good immune protection against the same type of enterovirus of the same serotype. Therefore, it is necessary and feasible to study the VLP vaccine of CVA6. • New findings: In this study, we developed a VLP vaccine against the dominant epidemic strain D3 genotype CVA6 causing hand, foot, and mouth disease in China, and evaluated its immune efficacy. We found that the levels of serum neutralizing antibodies obtained by different immunization methods were different, which was not mentioned in previous studies. meanwhile, both serum passive immunization and active immunization of VLP can protect neonatal mice from the fatal attack of the two evolutionary branches of D3a and D3b of CVA6.

Enterovirus D68 (EV-D68) is a significant global pathogen associated with severe respiratory infections and acute flaccid myelitis in children. Currently, there are no vaccines or antiviral drugs available for EV-D68, and a robust model to elucidate the pathogenesis of EV-D68 and evaluate treatment methods is lacking. We developed a mouse-adapted EV-D68 strain that caused progressive limb paralysis after intramuscular inoculation in 7-day-old mice. Viral load analysis showed that the skeletal muscle and spinal cord had the highest titers and most severe injuries. RNA sequencing of the infected muscle, brain, spinal cord, and lung tissues revealed differentially expressed genes (DEGs) associated with viral infection and pathogenesis. DEGs were significantly enriched in various pathways associated with antiviral immunity, interferon responses, and cytokine signaling. In the spinal cord, DEGs highlighted mitochondrial dysfunction and oxidative stress as crucial contributors to neural damage. Flow cytometry analysis of spinal cord cells showed that EV-D68 activates the immune system, leading to systemic inflammation and significant increases in CD8⁺ and CD4⁺ T cells, but limited neutrophil and monocyte infiltration. This mouse model provides a valuable tool for studying EV-D68 pathogenesis and evaluating antiviral and vaccine efficacy, thereby advancing the understanding of its neuropathological mechanisms. Importance: We developed a novel mouse model of EV-D68 that provides a valuable tool for studying its pathogenesis and evaluating antiviral and vaccine efficacy, deepening the understanding of its neuropathological basis.

Coxsackievirus A10 (CVA10) is an enterovirus that has caused global outbreaks of hand, foot, and mouth disease (HFMD), accompanied by neurological and systemic complications. Animal models that can simulate natural infections are necessary for studying viral pathogenesis. In this study, our objective was to establish a mouse-adapted strain (CVA10-P8) through serial passaging, which was capable of orally infecting 14-day-old ICR mice, leading to hind-limb paralysis and death. Viral titers in various tissues indicated tropism in muscle tissue, with significant increases observed in the brain, lung, and intestinal tissues as the infection progressed. Pathological examination revealed tissue damage in the muscles, brain, lungs, and intestines accompanied by neutrophil infiltration of the brain. Furthermore, flow cytometry and transcriptome analysis revealed metabolic abnormalities, immune system activation, and the promotion of systemic inflammatory responses in the brain, muscle, and lung tissues. In summary, we successfully developed a CVA10 mouse-adapted strain and a corresponding mouse model, providing valuable tools for studying CVA10 pathogenesis and evaluating the efficacy of antiviral interventions. IMPORTANCE: CVA10 has emerged as a predominant pathogen in the etiology of HFMD, with the potential to elicit neurological manifestations and systemic complications. In this study, we successfully established a novel murine model of CVA10 infection by serially propagating a clinical isolate of CVA10, which enabled oral infection in 14-day-old ICR mice. This model facilitated the investigation of the pathogenesis of CVA10-induced disease. Utilizing this infection model, we employed flow cytometry and transcriptome analysis to elucidate the central nervous system (CNS) inflammatory responses elicited by CVA10 in mice, which closely mimic the natural route of infection. Our findings provide novel insights into the pathophysiological mechanisms underlying CVA10-induced neuroinflammation and pave the way for further research into targeted therapeutic interventions for HFMD associated with CVA10.

Echovirus, a member of the Enterovirus B (EV-B) family, has led to numerous outbreaks and pandemics, causing a broad spectrum of diseases. Based on the national hand, foot, and mouth disease (HFMD) surveillance system, seven strains of echovirus 33 (E33) were isolated from Mainland of China between 2010 and 2018. The whole genomes of these strains were isolated and sequenced, and phylogenetic trees were constructed based on the gene sequences in different regions of the EV-B prototype strains. It was found that E33 may be recombined in the P2 and P3 regions. Five genotypes (A–E) were defined based on the entire VP1 region of E33, of which the C gene subtype was the dominant gene subtype at present. Recombinant analysis showed that genotype C strains likely recombined with EV-B80, EV-B85, E13, and CVA9 in the P2 and P3 regions, while genotype E had the possibility of recombination with CVB3, E3, E6, and E4. Results of Bayesian analysis indicated that E33 may have appeared around 1955 (95% confidence interval: 1945–1959), with a high evolutionary rate of 1.11 × 10⁻² substitution/site/year (95% highest posterior density (HPD): 8.17 × 10⁻³ to 1.4 × 10⁻² substitution/site/year). According to spatial transmission route analysis, two significant transmission routes were identified: from Australia to India and from Oman to Thailand, which the E33 strain in Mainland of China likely introduced from Mexico and India. In conclusion, our study fills the gaps in the evolutionary analysis of E33 and can provide important data for enterovirus surveillance.