Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed. Methods Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression. Results We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection. Conclusions Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Enterovirus Infection Restricts Long Interspersed Element 1 Retrotransposition

Long interspersed element 1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome that can serve as an endogenous upstream activator of cytoplasmic nucleic acid sensing pathways to elicit an antiviral immune response. In this study, we investigated the influence of enteroviral infection on L1 mobility. The results showed that infection with different enteroviruses, both EV-D68 and EV-A71, blocked L1 transposition. We screened diverse viral accessory proteins for L1 activity and identified EV-D68 2A, 3A, 3C, and EV-A71 ORF2p proteins as viral L1 inhibitors. EV-D68 2A suppressed L1 mobility by expression suppression of L1 proteins. Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins. The newly identified enteroviral protein ORF2p inhibited the expression of L1 ORF1p. Altogether, our findings shed light on the strict modulation of L1 retrotransposons during enterovirus replication.

Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

Sublethal Enteroviral Infection Exacerbates Disease Progression in an ALS Mouse Model

Background: Amyotrophic lateral sclerosis (ALS) is a fatalneurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors.Infection with enteroviruses, including poliovirus and coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALSdue to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathologicalphenotypes closely resembleALS. However, a causal relationship has not yet been affirmed.Methods:Wild-type C57BL/6J and SOD1G85R ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute and/or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting atweek 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression.Results: We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted inearly onset and progressive motor dysfunction, andshortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeksPI).Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TDP-43 pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, wediscovered that early (day 1) but not late (day 15) administration of ribavirincould rescue ALS-like neuropathology and symptoms induced by CVB3 infection.Conclusions: Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Clinical characteristics of severe neonatal enterovirus infection: a systematic review

Background Enterovirus (EV) is a common cause of infection in neonates. Neonates are at high risk of enterovirus infection with serious clinical manifestations and high lethality. This review systematically summarized the clinical characteristics of neonates with severe enteroviral infection to provide evidence for the identification and treatment of severe neonatal EV infection. Methods PubMed, Embase, and Web of Science were searched for original studies on neonates with severe EV infections from January 1, 2000, to November 27, 2020. Two reviewers independently screened the literature, extracted the data, and performed a descriptive analysis. Results In total, 66 articles with 237 cases of severe neonatal enterovirus infection were included. All neonates developed severe complications. Among them, 46.0% neonates had hepatitis or coagulopathy, 37.1% had myocarditis, 11.0% had meningoencephalitis, and 5.9% had other complications such as hemophagocytic lymphohistiocytosis and pulmonary hemorrhage. The lethality rate of neonates with severe infection was 30.4%. The highest lethality rate was 38.6%, which was observed in neonates with myocarditis. In 70.5% neonates, the age at the onset of symptoms was less than 7 days. Coxsackievirus B infection was seen in 52.3% neonates. The most common symptoms included temperature abnormalities (127, 53.6%), rash (88, 37.1%), poor feeding (58, 24.5%), and respiratory symptoms (52, 21.9%). The main treatment included transfusion of empirical antibiotics (127, 53.6%), blood components (100, 42.2%), intravenous immunoglobulin (IVIG; 97, 40.9%), mechanical ventilation (51, 21.5%), and extracorporeal membrane oxygenation (ECMO; 43, 18.1%). Additionally, antiviral medications pleconaril (14, 5.9%) and pocapavir (3, 1.3%) were administered. Conclusions Lethality was high in neonates with severe enterovirus infection, especially in those complicated with myocarditis. The most common symptoms included temperature abnormalities, rash, and poor feeding. The chief supportive treatment consisted of transfusion of blood components, mechanical ventilation, and ECMO. Empirical antibiotics and IVIG were widely used. Antiviral medications included pocapavir and pleconaril; however, more clinical evidence regarding their efficacy is needed.

Clinical Characteristics of Severe Neonatal Enterovirus Infection: A Systematic Review

Background: Enterovirus (EV) is a common cause of infection in neonates. Neonates are at high risk of enterovirus infection with serious clinical manifestations and high mortality. This review systematically summarized the clinical characteristics of neonates with severe enteroviral infection to provide evidence for the identification and treatment of severe neonatal EV infection. Methods: PubMed, Embase, and Web of Science were searched for original studies on neonates with severe EV infections from January 1, 2000, to November 27, 2020. Two reviewers independently screened the literature, extracted the data, and performed a descriptive analysis.Results: In total, 66 articles with 237 cases of severe neonatal enterovirus infection were included. All patients developed severe complications. Among them, 46.0% patients had hepatitis or coagulopathy, 37.1% had myocarditis, 11.0% had meningoencephalitis, and 5.9% had other complications such as hemophagocytic lymphohistiocytosis and pulmonary hemorrhage. The mortality rate of patients with severe infection was 30.4%. The highest mortality rate was 38.6%, which was observed in patients with myocarditis. In 70.5% patients, the age at the onset of symptoms was less than 7 days. Coxsackievirus B infection was seen in 52.3% patients. The most common symptoms included temperature abnormalities (127, 53.6%), rash (88, 37.1%), poor feeding (58, 24.5%), and respiratory symptoms (52, 21.9%). The main treatment included transfusion of blood components (42.2%), intravenous immunoglobulin (IVIG; 40.9%), mechanical ventilation (21.5%), and extracorporeal membrane oxygenation (ECMO; 18.1%). Additionally, antiviral medications pleconaril (14, 5.9%) and pocapavir (3, 1.3%) were administered. Conclusions: Mortality was high in patients with severe enterovirus infection, especially in those complicated with myocarditis. The most common symptoms included temperature abnormalities, rash, and poor feeding. The chief supportive treatment consisted of transfusion of blood components, mechanical ventilation, and ECMO. IVIG were widely used. Antiviral medications included pocapavir and pleconaril; however, more clinical evidence regarding their efficacy is needed.

Echovirus-30 Infection Alters Host Proteins in Lipid Rafts at the Cerebrospinal Fluid Barrier In Vitro

Echovirus-30 (E-30) is a non-polio enterovirus responsible for meningitis outbreaks in children worldwide. To gain access to the central nervous system (CNS), E-30 first has to cross the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BCSFB). E-30 may use lipid rafts of the host cells to interact with and to invade the BCSFB. To study enteroviral infection of the BCSFB, an established in vitro model based on human immortalized brain choroid plexus papilloma (HIBCPP) cells has been used. Here, we investigated the impact of E-30 infection on the protein content of the lipid rafts at the BCSFB in vitro. Mass spectrometry analysis following E-30 infection versus uninfected conditions revealed differential abundancy in proteins implicated in cellular adhesion, cytoskeleton remodeling, and endocytosis/vesicle budding. Further, we evaluated the blocking of endocytosis via clathrin/dynamin blocking and its consequences for E-30 induced barrier disruption. Interestingly, blocking of endocytosis had no impact on the capacity of E-30 to induce loss of barrier properties in HIBCPP cells. Altogether, these data highlight the impact of E-30 on HIBCPP cells microdomain as an important factor for host cell alteration.