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Travel-Associated Zika Cases and Threat of Local Transmission during Global Outbreak, California, USA

Zika and associated microcephaly among newborns were reported in Brazil during 2015. Zika has since spread across the Americas, and travel-associated cases were reported throughout the United States. We reviewed travel-associated Zika cases in California to assess the potential threat of local Zika virus transmission, given the regional spread of Aedes aegypti and Ae. albopictus mosquitoes. During November 2015–September 2017, a total of 588 travel-associated Zika cases were reported in California, including 139 infections in pregnant women, 10 congenital infections, and 8 sexually transmitted infections. Most case-patients reported travel to Mexico and Central America, and many returned during a period when they could have been viremic. By September 2017, Ae. aegypti mosquitoes had spread to 124 locations in California, and Ae. albopictus mosquitoes had spread to 53 locations. Continued human and mosquito surveillance and public health education are valuable tools in preventing and detecting Zika virus infections and local transmission in California.

Zika and associated microcephaly among newborns were reported in Brazil during 2015. Zika has since spread across the Americas, and travel-associated cases were reported throughout the United States. We reviewed travel-associated Zika cases in California to assess the potential threat of local Zika virus transmission, given the regional spread of Aedes aegypti and Ae. albopictus mosquitoes. During November 2015–September 2017, a total of 588 travel-associated Zika cases were reported in California, including 139 infections in pregnant women, 10 congenital infections, and 8 sexually transmitted infections. Most case-patients reported travel to Mexico and Central America, and many returned during a period when they could have been viremic. By September 2017, Ae. aegypti mosquitoes had spread to 124 locations in California, and Ae. albopictus mosquitoes had spread to 53 locations. Continued human and mosquito surveillance and public health education are valuable tools in preventing and detecting Zika virus infections and local transmission in California. The first human cases of Zika virus infection reported from the Americas were in May 2015 from Brazil (1). In the span of less than a year, Zika virus spread across South America, Central America, the Caribbean, and parts of Mexico. As observed with other mosquitoborne diseases, such as dengue and chikungunya, which have spread through Central and South America and the Caribbean, travel-associated cases of Zika were reported throughout the United States, and local transmission of Zika virus was eventually detected in Florida and Texas (2,3). Because California has established and expanding infestations of Aedes aegypti and Ae. albopictus mosquitoes, the main vectors of Zika virus, and is near Mexico, to which Zika virus is endemic, the risk for autochthonous transmission of Zika virus is a concern (4). During 2011–2015, Ae. aegypti and Ae. albopictus mosquitoes were detected in 85 cities and census-designated places in 12 counties of California (5). In California, patient testing and evaluation focused on assessment of infection in pregnant women and symptomatic patients, and assessment of potential viremia in these patients in relation to proximity to known Aedes mosquito infestations. To describe travel-associated Zika cases and better assess the potential threat of local Zika transmission in California, we reviewed all Zika cases reported to the California Department of Public Health (CDPH) during November 2015–September 2017. We also summarized surveillance for Ae. aegypti and Ae. albopictus mosquitoes in California and laboratory testing for Zika virus during this time. Zika cases were reported to CDPH by the 61 local health departments in California, either through the electronic California Reportable Disease Information Exchange (https://www.cdph.ca.gov/Programs/CID/DCDC/Pages/CalREDIE.aspx) or through paper case report forms. Cases reviewed by CDPH during November 2015–September 2017 were analyzed for type of Zika disease or infection, as defined by the 2016 Council of State and Territorial Epidemiologists (CSTE)/Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA) criteria and classified as confirmed or probable (6). Variables examined included sex, age, race/ethnicity, country where exposure likely took place, duration of travel, symptoms, symptom onset date, and pregnancy status and outcomes. We analyzed data by using SAS for Windows version 9.4 (SAS Institute Inc., Cary, NC, USA). For Zika case-patients with a travel duration of <6 months, we compared duration of time in Zika-affected areas between pregnant and all other case-patients by using the Kruskal-Wallis test for 2 groups (unequal variances) to retrospectively assess time at risk between these 2 groups. California has a network of local vector control agencies that monitors distribution and abundance of Aedes spp. and other mosquito populations. Mosquito surveillance typically includes trapping and identifying mosquitoes. Surveillance might be augmented by submitting mosquito specimens, especially specimens collected in and around residences or workplaces of case-patients, to the Davis Arbovirus Research and Training Laboratory at the University of California (Davis, CA, USA) for Zika virus (7), dengue virus (DENV) (8), and chikungunya virus (Davis Arbovirus Research and Training Laboratory at the University of California, unpub. data) testing by multiplex quantitative reverse transcription PCR (RT-PCR), as described by CDC (9). Mosquitoes submitted during West Nile virus (WNV) season (June 1–October 15) are also tested for WNV, St. Louis encephalitis virus, and western equine encephalitis virus (10). Agencies enter mosquito surveillance data into the California Vectorborne Disease Surveillance Gateway Database (https://gateway.calsurv.org/), which is used to generate statewide data for mapping of Aedes mosquito locations. We used a geographic information system (ArcGIS Desktop version 10.5; Esri, Redlands, CA, USA) to generate maps that enabled spatial and temporal mapping of Aedes mosquito populations in relation to presumed places of residence of presumed viremic Zika case-patients. We generated latitude and longitude data by using the Gateway Database for mosquitoes and determined case-patient place of residence by using the California Reportable Disease Information Exchange. Testing of humans for Zika virus was performed by the CDPH Viral and Rickettsial Disease Laboratory (VRDL), CDC, local public health laboratories, commercial laboratories, and blood banks. Testing for Zika virus infection was completed for appropriate tissue, serum, or urine specimens by using Zika virus nucleic acid or serologic tests. We analyzed symptomology and pregnancy status of those tested, volume of testing at the CDPH VRDL, types of tests conducted, and time from symptom onset to specimen collection date. For purposes of local transmission risk assessment, a potentially viremic patient was defined as a Zika-positive case-patient with symptom onset <7 days before or any time after return from travel to their place of residence. During November 2015–September 2017, a total of 588 travel-associated Zika cases were reported in California, including 139 infections in pregnant women, 10 congenital infections, and 8 sexually transmitted infections. Sixty-two case-patients were <18 years of age. On the basis of the 2016 CSTE surveillance case definition for Zika, 410 cases met the confirmed criteria and 178 were probable. Of these, 466 case-patients had noncongenital Zika disease with symptoms meeting the 2016 CSTE case definition for noncongenital Zika (>1 of the following: fever, rash, arthralgia, or conjunctivitis); 112 had a symptomatic noncongenital Zika infections, 6 had congenital Zika disease with Zika-associated birth defects (birth defects reported include those detected in infants infected with Zika virus before, during, or shortly after birth, including microcephaly, calcium deposits in the brain indicating possible brain damage, excess fluid in the brain cavities and surrounding the brain, absent or poorly formed brain structures, abnormal eye development, or other problems resulting from damage to the brain that affects nerves, muscles, and bones, such as clubfoot or inflexible joints, and confirmed hearing loss); and 4 had congenital Zika infections with no Zika-associated birth defects (6). A total of 66% (391/588) of case-patients were female; median age of case-patients was 35 years (range <1–89 years). Of persons with reported ethnicity, 69% (306/443) were Latino/Latina. For the 139 women pregnant at the time of diagnosis, median age was 27 years (range 14–44 years), and 78% (87/111) of those with reported ethnicity were Latina. Of 570 case-patients who contracted Zika virus while traveling outside California, most case-patients reported travel to Mexico (36.4%), Central America (34.3%), or the Caribbean (13.1%). The top 10 countries and territories for travel were Mexico (36.4%), Nicaragua (9.6%), Guatemala (8.4%), El Salvador (7.0%), Dominican Republic (4.4%), Puerto Rico (4.4%), Honduras (3.9%), Costa Rica (3.7%), Jamaica (2.5%), and Colombia (1.8%). The timeline for travel-associated Zika cases reported in California mirrored the spread of the outbreak across the Americas (Figure 1); the number of case-patients with travel to Mexico increased substantially starting in June 2016 as the number of Zika cases reported in Mexico steadily increased. Number of human Zika virus infections in residents, by month and year of onset and country of travel (top 10 countries shown), California, USA, October 1, 2015–September 1, 2017. Month was determined by date of symptom onset for symptomatic persons or specimen collection date for asymptomatic persons. Of 570 case-patients who traveled, 79 (13.9%) lived in their country of exposure for >6 months before coming to California, where they were subsequently tested for Zika virus. When we excluded these 79 persons, women who were pregnant at the time of Zika diagnosis had a significantly (p = 0.03) longer travel duration (median 14 days [range 1–153 days]) than all other Zika case-patients (median 11 days [range 1–137 days]). For 466 case-patients with symptoms, rash was the most common (89.0%, 415), followed by arthralgia (62.5%, 291), fever (60.1%, 280), myalgia (36.9%, 172), and conjunctivitis (35.0%, 161). A rash without any other symptom was seen in 49 (10.5%) case-patients. For those case-patients with >1 symptom, the most common combination of symptoms, reported by 13% of case-patients, was rash, arthralgia, and fever. Seven case-patients were hospitalized for a median of 3 (range 1–8) days. On the basis of symptom onset date, the number of Zika cases reported in California in 2016 increased from June through August and then decreased through November (Figure 2). Confirmed and probable symptomatic Zika virus infections, by symptom onset month and year, California, USA, October 2015–September 2017. Of 139 women who were pregnant at the time of Zika diagnosis, 120 had completed their pregnancies by September 1, 2017: 114 with live births and 6 with fetal losses. Fourteen women were still pregnant, and the status of 5 women was unknown. For live births, 90 (78.9%) infants were tested for Zika virus at or shortly after birth; 84 (73.7% of live births) infants showed negative results for Zika virus by nucleic acid and IgM tests, and 6 (5.3% of live births) showed positive results. Of the remaining 24 live births, 7 infants were not tested, and the testing status of 17 infants was unknown. In addition to the 6 congenitally infected infants that were born to Zika virus–positive mothers, 4 additional infants whose mothers were exposed to Zika virus but showed negative results by nucleic acid or IgM tests were positive for Zika virus. Eight infants were born in California with Zika-associated birth defects. Of these infants, 2 were negative and 6 were positive for Zika virus by PCR and IgM test. Both Zika virus–negative infants had mothers who were positive for Zika virus, and 3 of the Zika virus–positive infants had mothers who had negative results for Zika virus. During January 1, 2016–September 1, 2017, we detected 78 new locations for Ae. aegypti mosquitoes and 25 new locations for Ae. albopictus mosquitoes, for a total of 133 cities or census-designated places for Ae. aegypti mosquitoes and 56 for Ae. albopictus mosquitoes, an increase of 142% for Ae. aegypti mosquitoes and 81% for Ae. albopictus mosquitoes in 20 months. In 2017, Ae. aegypti mosquitoes were detected in 12 counties and Ae. albopictus mosquitoes in 5 counties, including 2 new counties containing Ae. aegypti mosquitoes in the Central Valley (11). As of September 1, 2017, a total of 13,499 Ae. aegypti mosquitoes and 2,719 Ae. albopictus mosquitoes had been tested by Davis Arbovirus Research and Training for Zika virus, chikungunya virus, and DENV. None of these mosquitoes were positive for these arboviruses, although 5 pools of Ae. aegypti mosquitoes and 1 pool of Ae. albopictus mosquitoes were positive for WNV. Of the 588 case-patients reported who had Zika virus infections, 435 (74.6%) were likely viremic while in California. Of those viremic case-patients, 279 (64.1%) were also residents of California counties where Ae. aegypti or other Aedes spp. mosquitoes have been detected; their co-location was more common in southern California (Figure 3). Locations where Aedes spp. mosquitoes were detected and residences of possibly viremic case-patients infected with Zika virus, central (A) and southern (B) California, USA, October 2015–September 2017. Insets show larger views of corresponding region. Although the VRDL performed most (58.7%; 345/588) testing for Zika-positive cases in California, commercial laboratories accounted for 17.5% (103/588), local health departments for 13.1% (77/588), and CDC for 9.5% (56/588). Seven Zika cases reported in California were identified through blood bank screening. Most testing at VRDL was performed for asymptomatic pregnant women (7,795 asymptomatic pregnant women/11,603 total patients; 67.2%). Eighty (1.0%) of these asymptomatic pregnant women were positive for Zika virus by quantitative RT-PCR (1 woman) or IgM test and plaque-reduction neutralization test (PRNT) (79 women). Of the 120 completed pregnancies for women who were infected with Zika virus while pregnant, 45 placental tissues (including placenta, membrane, and umbilical cord) were sent to CDC for testing. Zika virus was detected by RT-PCR in placental tissues of 8 women. Detection of Zika virus in the placental tissue provided confirmatory testing for 5 of these women (3 were already confirmed by serum PRNT). Of the 410 confirmed Zika cases reported in California, 319 (77.8%) case-patients had Zika virus detected by nucleic acid tests in serum, urine, or placental tissue, and the other 91 were confirmed by detection of neutralizing antibodies to Zika virus (and not DENV). For symptomatic case-patients, the median time from illness onset to specimen collection was 5 days (range 1–194 days). For cases confirmed by serum or urine nucleic acid tests, the time to collection was shorter, with a median of 3 days (range 1–33 days), than for PRNT, with a median of 16 days (range 1–194 days). Since the global Zika outbreak began in South America in 2015, many travel-associated Zika cases have been documented in California, including infections in pregnant women, congenital infections, and sexually transmitted infections. With the establishment and continuing spread of Ae. aegypti and Ae. albopictus mosquitoes in California, prevention of local transmission of Zika virus has been and continues to be a public health priority. In working to identify possible local transmission, CDPH used the data for travel-associated Zika cases described in this article to develop our Zika testing prioritization. Although CDC recommended specific criteria for travel-associated Zika virus testing, different criteria were needed when testing persons without travel history, especially when the number of confirmed Zika cases was increasing in California and local Zika virus transmission was reported in Florida (2). The goal of such testing was to identify anyone who potentially had Zika virus, without testing large numbers of persons at low risk. CDPH subsequently provided criteria for local health departments in California to consider in evaluating whether suspected persons without travel history should be considered for Zika virus testing, including factors that could increase risk for local transmission, as well as signs and symptoms most suggestive of Zika. For example, CDPH allowed that, for counties where Aedes mosquitoes have been detected, Zika virus testing could be offered to persons who live in an area containing Aedes spp. mosquitoes and who came to their healthcare provider with a maculopapular rash and 1 other symptom consistent with Zika (fever, arthralgia, or conjunctivitis), without an alternative explanation, such as a drug reaction or other infection. Rash was recommended as the primary criterion in this setting because nearly 90% of Zika case-patients had a rash. This allowance for Zika virus testing for persons with no travel or sexual exposure was used in some counties in California and identified several persons suspected of having Zika who were tested, all of whom showed negative results. This testing allowance would not be appropriate in areas that did not contain Aedes spp. mosquitoes and is being reconsidered as the number of Zika cases has decreased. Although California health officials did not identify any episodes of local Zika virus transmission, our data indicate that large numbers of likely viremic travelers returned to areas containing Ae. aegypti and Ae. albopictus mosquitoes, especially in southern California, as has also been found for dengue and chikungunya (4). This overlap of viremic travelers and Aedes spp. mosquito vectors potentially increases the risk for local transmission and will continue to be a public health concern requiring ongoing mosquito and human case surveillance. CDPH works closely with local health departments and vector control agencies to prepare for the potential of a locally transmitted outbreak. The close coordination of mosquito control programs in California with programs of local health departments, the common use of air conditioning or window screens by residents, and the variable distribution of Aedes spp. mosquitoes in some affected counties in California would likely limit the extent of a local outbreak should it occur. Nonetheless, knowledge of co-located vector mosquitoes and infected returned travelers is needed to aid in rapid investigation of any suspected locally transmitted case(s) and to limit potential spread. Mosquito seasonality influences risk for local transmission, and although Aedes spp. mosquitoes can be found year-round in California, they are most abundant from June through November, typically peaking in September and October (5). Large numbers of potentially viremic case-patients returned to California during June–November 2016 (Figure 2), corresponding with the season of high Aedes spp. mosquito activity in California. This seasonality also reflects Ae. aegypti mosquito activity in northern Mexico, where Ae. aegypti mosquitoes are abundant from August through November. Similar to the chikungunya outbreak in the Americas that began in 2013 and rapidly peaked in most locations before decreasing (12), the number of Zika cases is now decreasing. This decrease in Zika cases has been observed both in countries reporting local transmission and in the number of infected returned travelers reported in the United States and in California (13). Although the level of Zika virus transmission has decreased, many countries, including Mexico, have continued to report moderate levels of local Zika virus transmission (14). Given the large number of travelers between Mexico and California, it is critical that Zika prevention messaging, surveillance, and outreach continue, especially as it pertains to women traveling while pregnant. The large volume of testing for asymptomatic pregnant women reinforces that potential Zika virus exposure incidents were occurring in high numbers even with extensive provider education and public health messaging in California and nationally. Women who were pregnant at the time of their Zika diagnosis had a longer duration of travel in their exposure country than all other case-patients. Because most infected pregnant women were Latina, it is possible that many of them had traveled to visit family and therefore had longer stays. Given the health risk to pregnant women and their fetuses, this finding is of great concern. We need to ensure that English- and Spanish-language public health messaging about risks of travel or travel of sexual partners to Zika-affected countries continues to reach pregnant women and their healthcare providers. Although a decrease in reported travel-associated Zika cases was observed in California in March 2017, we did not detect a decrease in specimens submitted for asymptomatic pregnant women to VRDL until August 2017. Laboratory testing for Zika virus has proved challenging throughout the outbreak. Results of assays were difficult to interpret because serologic cross-reactivity with other flaviviruses, especially DENV, was common (15). Detection of neutralizing antibodies against Zika virus and DENV was observed for 178 probable Zika case-patients reported in California. Thus, the specific flavivirus of the infection in these case-patients could not be determined. In addition, Zika IgM has been reported to persist in serum, making timing of infection difficult to determine (16). The discordant testing results observed in the mother/infant pairs were equally challenging, suggesting that a negative test result could rarely rule out a Zika virus infection. All these factors, in addition to the difficulty of determining the date of exposure for many case-patients, especially for women who lived in the area of exposure for an extended time, made the interpretation of negative results problematic and created challenges for ensuring that affected infants received appropriate follow-up care. Our study and data interpretation have several limitations. First, the data included only case-patients who were positive for Zika virus, not case-patients who were negative but had been potentially exposed to Zika virus. Analysis of such persons who were negative for this virus but had potentially been exposed would be helpful to further delineate risk and discriminate symptoms. However, negative results, particularly from commercial laboratories, often have limited associated clinical and demographic data. Second, some dengue cases might be misclassified as Zika cases because of cross-reactivity and nonspecific binding in available serologic assays. Given the large percentage of case-patients in California with previous exposure to flaviviruses, especially DENV, there is potential for false-positive interpretation of PRNT results. All case-patients with neutralizing antibodies against DENV and Zika virus were classified on the basis of the CSTE case definition as having Zika because of the higher risk during pregnancy from exposure to Zika virus. In addition, low pretest probability, especially in asymptomatic persons, increases the risk for misclassification because of type I errors (false-positive results). Third, an estimated 80% of persons infected with Zika virus are asymptomatic (17), making it difficult to determine when, where, and how many potentially viremic persons are returning to California. Fourth, there is a clear testing bias toward pregnant and reproductive-age women, which skews demographic data. Although Zika virus transmission and Zika case numbers have decreased across the Americas, we expect to see continued, limited, local transmission in some affected countries. Thus, there is still a risk for pregnant women and all those who travel to these countries, and it is necessary that prevention messaging remains targeted and operative. Healthcare providers should continue to be suspicious of returning travelers with rash, fever, conjunctivitis, or arthralgia, particularly when other diagnoses have been ruled out. The expansion of Ae. aegypti and Ae. albopictus mosquitoes into 12 counties in California, especially along the southern border region, increases the risk for local Zika transmission in California. The large percentage of potentially viremic travelers returning to areas that contain Aedes spp. mosquitoes, in addition to an unknown number of returned travelers who are asymptomatically infected but not detected, makes the risk for local transmission a continuing threat, albeit low, in California. Zika has complicated disease manifestations and transmission dynamics, such as sexual and congenital transmission, which are not typically observed for other arboviruses. It is vital that we apply the public health lessons learned during the Zika outbreak to prepare for complexities that might arise during future epidemics of emerging and reemerging arboviruses. We thank the Viral and Rickettsial Disease Laboratory at CDPH; the Center for Family Health at CDPH, particularly Karen Ramstrom; the Centers for Disease Control and Prevention; the University of California, Davis, particularly Christopher Barker; and local California health departments and vector-control agencies for their collaborative efforts. We also thank Marco Metzger and Greg Hacker for providing Aedes spp. mosquito surveillance data and mapping expertise and Anne Kjemtrup for detailed manuscript review. This study was supported by the Epidemiology and Laboratory Capacity for Infectious Diseases Cooperative Agreement no. 6 NU50CK000410 from the Centers for Disease Control and Prevention. Suggested citation for this article: Porse CC, Messenger S, Vugia DJ, Jilek W, Salas M, Watt J, et al. Travel-associated Zika cases and threat of local transmission during global outbreak, California, USA. Emerg Infect Dis. 2018 Sep [date cited]. https://doi.org/10.3201/eid2409.180203 Dr. Porse is an epidemiologist in the Vector-Borne Disease Section, Infectious Diseases Branch, Division of Communicable Disease, California Department of Public Health, Sacramento, CA. Her research interests include epidemiology, mosquitoborne diseases, and public health.

9 2018

Emerging Infectious Diseases

10.3201/eid2409.180203

Increased functional connectivity of amygdala subregions in patients with drug-naïve panic disorder and without comorbidities

Amygdala plays an important role in the neurobiological basis of panic disorder (PD), and the amygdala contains different subregions, which may play different roles in PD. The aim of the present study was to examine whether there are common or distinct patterns of functional connectivity of the amygdala subregions in PD using resting-state functional magnetic resonance imaging and to explore the relationship between the abnormal spontaneous functional connectivity patterns of the regions of interest (ROIs) and the clinical symptoms of PD patients. Fifty-three drug-naïve, non-comorbid PD patients and 70 healthy controls (HCs) were recruited. Seed-based resting-state functional connectivity (rsFC) analyses were conducted using the bilateral amygdalae and its subregions as the ROI seed. Two samples t test was performed for the seed-based Fisher's z-transformed correlation maps. The relationship between the abnormal spontaneous functional connectivity patterns of the ROIs and the clinical symptoms of PD patients was investigated by Pearson correlation analysis. PD patients showed increased rsFC of the bilateral amygdalae and almost all the amygdala subregions with the precuneus/posterior cingulate gyrus compared with the HC group (left amygdala [lAMY]: t = 4.84, P <0.001; right amygdala [rAMY]: t = 4.55, P <0.001; left centromedial amygdala [lCMA]: t = 3.87, P <0.001; right centromedial amygdala [rCMA]: t = 3.82, P = 0.002; left laterobasal amygdala [lBLA]: t = 4.33, P <0.001; right laterobasal amygdala [rBLA]: t = 4.97, P <0.001; left superficial amygdala [lSFA]: t = 3.26, P = 0.006). The rsFC of the lBLA with the left angular gyrus/inferior parietal lobule remarkably increased in the PD group (t = 3.70, P = 0.003). And most of the altered rsFCs were located in the default mode network (DMN). A significant positive correlation was observed between the severity of anxiety and the rsFC between the lSFA and the left precuneus in PD patients (r = 0.285, P = 0.039). Our research suggested that the increased rsFC of amygdala subregions with DMN plays an important role in the pathogenesis of PD. Future studies may further explore whether the rsFC of amygdala subregions, especially with the regions in DMN, can be used as a biological marker of PD.

Amygdala plays an important role in the neurobiological basis of panic disorder (PD), and the amygdala contains different subregions, which may play different roles in PD. The aim of the present study was to examine whether there are common or distinct patterns of functional connectivity of the amygdala subregions in PD using resting-state functional magnetic resonance imaging and to explore the relationship between the abnormal spontaneous functional connectivity patterns of the regions of interest (ROIs) and the clinical symptoms of PD patients. Fifty-three drug-naïve, non-comorbid PD patients and 70 healthy controls (HCs) were recruited. Seed-based resting-state functional connectivity (rsFC) analyses were conducted using the bilateral amygdalae and its subregions as the ROI seed. Two samples t test was performed for the seed-based Fisher's z-transformed correlation maps. The relationship between the abnormal spontaneous functional connectivity patterns of the ROIs and the clinical symptoms of PD patients was investigated by Pearson correlation analysis. PD patients showed increased rsFC of the bilateral amygdalae and almost all the amygdala subregions with the precuneus/posterior cingulate gyrus compared with the HC group (left amygdala [lAMY]: t = 4.84, P <0.001; right amygdala [rAMY]: t = 4.55, P <0.001; left centromedial amygdala [lCMA]: t = 3.87, P <0.001; right centromedial amygdala [rCMA]: t = 3.82, P = 0.002; left laterobasal amygdala [lBLA]: t = 4.33, P <0.001; right laterobasal amygdala [rBLA]: t = 4.97, P <0.001; left superficial amygdala [lSFA]: t = 3.26, P = 0.006). The rsFC of the lBLA with the left angular gyrus/inferior parietal lobule remarkably increased in the PD group (t = 3.70, P = 0.003). And most of the altered rsFCs were located in the default mode network (DMN). A significant positive correlation was observed between the severity of anxiety and the rsFC between the lSFA and the left precuneus in PD patients (r = 0.285, P = 0.039). Our research suggested that the increased rsFC of amygdala subregions with DMN plays an important role in the pathogenesis of PD. Future studies may further explore whether the rsFC of amygdala subregions, especially with the regions in DMN, can be used as a biological marker of PD. Panic disorder (PD) is a common anxiety disorder, which is manifested by recurrent unexpected panic attacks, along with concerns about additional panic attacks or behavioral changes related to the panic attacks.[1] PD is associated with diminished emotional health, decreased social functioning, and lower quality of life.[2] Although PD is a prevalent and debilitating disorder, its neuronal bases have not been fully determined. Based on a popular hypothesis proposed by Gorman et al,[3] the amygdala is a core brain region in fear network, and the function of the amygdala and its functional interaction with both cortical and subcortical structures (e.g., medial prefrontal cortex, hippocampus, hypothalamus, and brain stem) account for the physiological and behavioral response to the conditioned fear stimulus and panic attack. Evidence from neuroimaging studies further indicates the role of the amygdala in the pathophysiology of PD.[4] For example, the hyper-activation of the amygdala occurs in patients with PD during processing conditioned fear, and perception of negative picture or facial expression.[5–7] In humans, the amygdala can be divided into three subregions, namely, the centromedial amygdala (CMA), basolateral amygdala (BLA), and superficial amygdala (SFA).[8] Each of these subregions has independent connectivity profiles with other regions, thus displaying distinct functions.[9] The distinct connectivity profiles of the amygdala subregions and their abnormalities have been found in individuals with high trait anxiety,[10] major depressive disorder,[11] and obsessive-compulsive disorder.[12] PD patients showed increasing activation in the right central and basolateral amygdala during the phasic response task when compared with healthy controls (HCs).[7] All these researches indicate the independent roles of the amygdala subregions to some extent. For now, no study has investigated the functional roles that different subregions of the amygdala may play in PD. Thus, it is not clear whether there are different changes of the rsFC of amygdala subregions in PD. Therefore, it is interesting to investigate whether there are subregional specificities of the amygdala connectivity in PD. In brief, the resting-state functional connectivity (rsFC) of the amygdala subregions with other brain region has not been previously studied in PD patients. Therefore, the current study aimed to investigate the differences in the rsFC of the amygdala and its subregions between medication-naïve PD patients without comorbidities and HCs through resting-state functional magnetic resonance imaging (rs-fMRI) analysis. Besides, we further investigated the relationship between the functional connectivity patterns, which were different between PD and HC, and the clinical symptoms of PD patients. Considering the heterogeneity of the amygdala, we hypothesized that there are specific rsFCs of the different amygdala subregions with other brain regions in PD patients. This study was approved by the Research Ethics Committee of the Beijing Anding Hospital, Capital Medical University (2020 scientific research No. 31), and conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants before the study. Sixty right-handed patients with PD (33 females and 27 males) were recruited from the Outpatient Department of Beijing Anding Hospital, Capital Medical University. The diagnosis of PD patients was made by two trained psychiatrists, using the Mini International Neuropsychiatric Interview (MINI) 5.0.[13] The PD inclusion criteria were: (1) patients who met the Diagnostic and Statistical Manual of Mental Disorders criteria of PD, with other psychiatric disorders excluded such as schizophrenia, bipolar disorders, depression, and agoraphobia (determined by senior psychiatrists using the MINI 5.0); (2) patients aged between 18 and 60 years; (3) score >7 on the Panic Disorder Severity Scale-Chinese version (PDSS-CV), and <18 on the 17-item Hamilton Depression Rating Scale (HAMD-17); (4) no contraindications to magnetic resonance imaging (MRI) scanning; (5) no history of neurological illnesses; (6) those who received at least primary school education and understood the scales; and (7) patients who had no history of exposure to anti-depressants, psychotherapy, or physical therapy. Three patients were excluded because of using benzodiazepines before the MRI scan. We also recruited 71 healthy volunteers (29 females and 42 males, aged between 19 and 54 years). None of the participants in the HCs had any history of psychiatric disorders (screened using the MINI 5.0), and any significant neurological illness such as epilepsy, encephalitis, Parkinson's disease, tumors, ischemia, and hematomas. All participants in the HCs were not pregnant, and were without contraindications for MRI scanning according to self-reports. The Panic Disorder Severity Scale was compiled by Shear et al[14] in 1997, and the Chinese version (PDSS-CV) was revised by one of the authors of the current study. According to the validation results, the PDSS-CV has good validity and reliability in Chinese population, the internal consistency reliability was 0.83, the total test-retest correlation coefficient was 0.95, the test-retest coefficient of each item was between 0.72 and 0.95, and the intraclass correlation coefficient among raters was 0.94.[15] PDSS-CV was a clinician-rated scale of PD severity, which contains items that can assess the severity of panic disorder and associated symptoms from seven dimensions: (1) frequency of panic attacks; (2) distress during panic attacks; (3) anticipatory anxiety (worry about future panic attacks); (4) agoraphobic fear and avoidance; (5) interoceptive fear and avoidance (i.e., apprehension and avoidance of bodily sensations); (6) impairment or interference in work functioning; and (7) impairment or interference in social functioning. The scale was administered by a clinician using a scripted interview. And all indexes were assessed based on the conditions of the last month. The PDSS-CV cut-off scores for severity of PD were as follows: score 8 to 10 was slight; 11 to 13 was moderate; 14 to 16 was marked, and 17 and above was serious. The HAMD-17 and Hamilton Anxiety Rating Scale (HAMA) were used to measure the severity of depressive and anxiety symptoms during the past week.[16,17] MRI data were acquired using a 3.0 T MRI scanner (Prisma, Siemens, Erlangen, Germany) with a 64-channel phased-array head coil at the Radiology Department of Beijing Anding Hospital, Capital Medical University. Foam paddings were used to minimize head movement, and earplugs were used to minimize scanner noise. Structural and functional images were acquired for each subject. All subjects were instructed to lay in a supine position with eyes closed, but not to fall asleep or think of anything deliberately. Functional images were obtained using an echo-planar imaging sequence with the following parameters: repetition time (TR) = 2000 ms, echo time (TE) = 30.00 ms, flip angle = 90°, matrix = 64 × 64, the field of view (FOV) = 200 mm × 200 mm, number of slices = 33, thickness/gap = 3.50/0.70 mm, and voxel size = 3.13 mm × 3.13 mm × 4.20 mm. A total of 200 volumes of rs-fMRI were collected for each participant. High-resolution sagittal T1 images were acquired using the 3D magnetization-prepared rapid gradient-echo sequence with the following parameters: TR = 2530 ms, TE = 1.85 ms, inversion time (TI) = 800 ms, flip angle = 15°, FOV = 256 mm × 256 mm, slices number = 192, thickness = 1.00 mm, no gap, and voxel size = 1.00 mm × 1.00 mm × 1.00 mm. The fMRI data were preprocessed using the Data Processing and Analysis for Brain Imaging (DPABI_V6.0, https://rfmri.org/DPARSF)[18] software based on the Statistical Parametric Mapping (SPM12, http://www.fil.ion.ucl.ac.uk/spm/). The preprocessing procedures included the following steps: removal of the first five time points; slice timing correction; realignment; nuisance variable regression; normalization (T1 images were used); resampling to a resolution of 3 mm × 3 mm × 3 mm,[18] spatial smoothing (6 mm full-width at half-maximum Gaussian kernel), and band-pass filtering (0.01–0.10 Hz). The nuisance covariates included the Frison's 24 motion parameters, white matter signals, cerebral spinal fluid signals, and the headmotion scrubbing regressors.[19,20] In order to avoid artificial negative correlations in rsFC analysis, the global signals were not included in nuisance covariates regression.[21] We limited our data analysis to participants with a receivable range of head motion to reduce the effect of motion-related artifacts on rsFC. First, we used volume-based frame-wise displacement (FD) to quantify micro head motions.[22] In this study, the threshold for FD value was 0.2 mm. Participants who had more than 100 volumes, which exceed the threshold, were excluded.[23] Second, participants with obvious head motions (>2° of maximal rotation or >2 mm of maximal translocation) were excluded. A total of four PD patients and one HC were excluded. Finally, we used the mean FD as a covariate in group-level analyses to further reduce the potential effect of micro head motion on the rsFC. The seeds of amygdala subregions (e.g., CMA, BLA, and SFA) were derived from histological properties of the amygdala based on the cytoarchitectonic assessment of 10 human postmortem brain.[24,25] According to previous studies,[26–30] we used maximum probability maps to keep each voxel non-overlapping and assign each voxel exclusively to a single region. The subregion masks were resampled totally to generate resolution of 3 mm × 3 mm × 3 mm, and then taken as our seed regions for rsFC analyses [Figure 1].[12] The specific location of the subregions of the amygdala by using the Statistical Parametric Mapping Anatomy toolbox. Blue: Right superficial amygdala; Dark blue: Right centromedial amygdala; Green: Left basolateral amygdala; Red: Left centromedial amygdala; Violet: Right basolateral amygdala; Yellow: Left superficial amygdala. The seed-based rsFCs of the bilateral amygdala and its six subregions with other brain regions were analyzed. By averaging the time series of all voxels within the seed region, the mean time series of each seed region was acquired. Pearson's correlation coefficients were computed between the mean time series of the seed region and the time series of each voxel of the whole brain. The correlation coefficients were converted into z values by using Fisher's r-to-z transformation to improve their normality. These z score maps were then used to conduct second-level analysis. In order to verify the results, we also conducted a flexible factorial analysis to further test the specificity of abnormal functional connectivity of the amygdala subregions with other brain regions. Different statistical methods were used to assess the differences in demographic data between the PD and the HCs. Normally distributed variables were presented as mean ± standard deviation and analyzed by two-sample t tests, and non-normally distributed variables were presented as median (Q1, Q3) and analyzed using Mann–Whitney U test. Categorical variables were presented as number (percentage) and analyzed using chi-squared test or Fisher's exact test. One-sample t test was performed to exhibit the rsFC patterns of the seed region within each group (voxel-level P <0.001, cluster-level family wise error [FWE] corrected P <0.05). A two-sample t test was performed to identify brain regions, which have inter-group differences in connectivity with the seed region, when adjusting for the confounding effects of the age, gender, education level, and mean FD. Besides, in order to identify whether those regions were located in the Default Network Mode (DMN), we calculated the intersection set of these regions with the Yeo's DMN template.[31] The statistical threshold of the two-sample t test was set at a voxel-level P <0.001 in combination with a cluster-level FWE Pcorrected <0.00625 to correct for multiple comparisons. Because the rsFC analysis was repeated eight times for the number of independent seed regions, we used Bonferroni correction for multiple comparisons (Pcorrected <0.00625 = 0.05/8). According to the result of FC comparison between the PD group and the HCs, we determined the regions with increased FC in the PD group and extracted the FC value in each region of the PD group. A Pearson's correlation analysis was carried out to assess the association between the strength of FC and the severity of symptoms (indicated by HAMA or PDSS-CV). The statistical threshold was set as P <0.05. We finally included 53 PD patients and 70 healthy volunteers in this study. The clinical and demographic data are shown in Table 1. No significant differences were observed in age, gender, education, and head motion between the PD and HC groups. The mean PDSS-CV score of the PD patients was 12.15 ± 2.44. The HAMA and HAMD-17 score of the PD patients was 13 (12, 18) and 9 (6, 13), respectively. All PD patients who were included in the analysis have not been treated with any medication or psychotherapy before the study. Demographic and clinical characteristics of patients with panic disorder and healthy controls. Data are presented as mean ± standard deviation, median (Q1, Q3) or n (%). t values. χ2 values. U values. FD: Frame-wise displacement; HAMA: Hamilton anxiety rating scale; HAMD-17: 17-item hamilton depression rating scale; HC: Healthy control; PD: Panic disorder; PDSS-CV: Chinese version of panic disorder severity scale; –: Not applicable. We separately used the bilateral amygdalae and each subregion as a seed region to reveal the rsFC patterns of each region. The brain areas with positive rsFC of the bilateral amygdalae and its six subregions were concentrated mainly in the frontal gyrus, temporal gyrus, insula, anterior cingulate cortex, occipital gyrus, and fusiform gyrus in both the HC and PD groups (cluster-level FWE Pcorrected <0.05). In the HC group, we observed negative rsFC in the right amygdala (rAMY), the left centromedial amygdala (lCMA), the right centromedial amygdala (rCMA), and the right superficial amygdala (rSFA) with the precuneus (PCu), inferior parietal lobule, and middle frontal gyrus. And, in the PD group, we observed negative rsFC in the lCMA, the left laterobasal amygdala (lBLA), the left superficial amygdala (lSFA), and rSFA with the middle frontal gyrus (cluster-level FWE Pcorrected <0.05) [Figure 2]. Spatial distributions of the rsFC of the eight seed regions of the HC and PD group. The images were created using BrainNet Viewer (https://www.nitrc.org/projects/bnv/). HC: Healthy control; lAMY: Left amygdala; lBLA: Left laterobasal amygdala; lCMA: Left centromedial amygdala; lSFA: Left superficial amygdala; PD: Panic disorder; rAMY: Right amygdala; rBLA: Right laterobasal amygdala; rCMA: Right centromedial amygdala; rSFA: Right superficial amygdala; rsFC: Resting-state functional connectivity. Based on the comparison of the rsFC patterns between the two groups, PD patients showed increased rsFC between the bilateral amygdalae and the PCu as well as the posterior cingulate cortex (PCC) compared with the HC group (cluster-level FWE Pcorrected <0.006). Additionally, compared with the HC group, all the amygdala subregions (except rSFA) showed increased positive rsFC with the PCu in the PD group (cluster-level FWE Pcorrected <0.006) [Table 2]. Moreover, compared with the HC group, increased rsFC was observed between the lBLA and the left angular gyrus/inferior parietal lobule in the PD group (cluster-level FWE Pcorrected <0.006) [Table 2]. We did not find significant group differences in the rsFC of the rSFA. We calculated the intersection of the brain regions, which have the increased rsFC in the amygdala or its subregions in PD patients, with the DMN in the Yeo's network template, and verified that most of regions were located in the DMN [Supplementary Figures 1 and 2]. Brain regions with increasing rsFC in the amygdala and its subregions in PD patients compared with HC. BA: Brodmann area; FC: Functional connectivity; FWE: Family wise error; HC: Healthy control; lAMY: Left amygdala; lBLA: Left laterobasal amygdala; lCMA: Left centromedial amygdala; lSFA: Left superficial amygdala; MNI: Montreal neurological institute; PCu/PCC: Precuneus/posterior cingulate cortex; PD: Panic disorder; rAMY: Right amygdala; rBLA: Right laterobasal amygdala; rCMA: Right centromedial amygdala; rSFA: Right superficial amygdala; rsFC: Resting-state functional connectivity; SD: Standard deviation; –: Not available. We performed a flexible factorial analysis with group (PD vs. HC) as one independent factor and subregion as the repeated measurement factors by SPM. The group (PD vs. HC) and subregions (lCMA vs. lBLA vs. lSFA vs. rCMA vs. rBLA vs. rSFA) were considered as the main effects, the group × subregions was taken as the interaction effect, and the factors of age, gender, education, and head motion were taken as covariates. We found group and subregion were with main effects. However, no significant interaction effect was observed between group and subregions (voxel-level P <0.001, cluster-level FWE P <0.05) [Supplementary Figure 3 and Supplementary Table 1] No significant correlations were observed between the rsFC in the amygdala or its subregions and the PDSS-CV scores and the course of PD. However, the HAMA score of PD patients was positively correlated with the strength of the rsFC between the lSFA and the left PCu (P <0.05) [Figure 3]. Correlations between anxiety severity (measured by HAMA) and the rsFC of lSFA with the left precuneus in the PD group. HAMA: Hamilton Anxiety Rating Scale; lSFA: Left superficial amygdala; PD: Panic disorder; rsFC: Resting-state functional connectivity. The results showed that there are hyper-connectivities between the PCu and the bilateral amygdalae and its subregions (except the rSFA) in PD group compared with HC. The PCu is closely related to the monitoring of the internal and external environment, maintenance of consciousness, processing of emotions, self-introspection, and the extraction of episodic memory.[32] The PCu is constantly engaged in information gathering and representation of the self and the external world.[33] Worry is associated with increasing activation in the PCu.[34] Panic initiation effect in PD patients is reflected by the enhanced activation of the PCC and the anterior cingulate cortex.[35] The increased connectivities between the amygdala and the PCu/PCC found in our study validated the previous finding which was obtained in a smaller sample of PD patients (n = 11) who had used medication.[36] More importantly, our findings were obtained in non-comorbid PD patients, which can exclude the effects of comorbidity. We also found that the rsFC between the lBLA and the left angular gyrus/inferior parietal lobule was increased in PD group compared with HC group, which was a specific change among amygdala subregions. Angular gyrus/inferior parietal lobule are associated with bodily self-consciousness.[37] The BLA participates in the information processing and integration related to self-cognition.[38] The increased rsFC between the lBLA and the left angular gyrus/inferior parietal lobule may be related to the enhanced internal sensibility and the inability to reasonably adjust the self-cognition of physical discomfort in PD patients, which ultimately leads to the deviant cognition. Interestingly, most of the brain regions, which have increased rsFC in the amygdala and its subregions, were located in the DMN. The DMN involves in self-referential processing.[39] A meta-analysis study found that the DMN is associated with affective and safety cue learning,[40] which was less noticed.[41] A study carried out in healthy volunteers also confirmed that the functional connectivity between the DMN and the amygdala is related to the cue condition during fear and anxiety learning, and it was described as a facilitating state of readiness in response to environmental changes.[42] Although the findings of abnormal DMN activity in patients with PD were inconsistent,[43–45] the DMN may play a central role in the pathogenesis of panic disorder. By combining our results with the previous findings, we speculated that the hyper-connectivity between the amygdala or its subregions with the DMN, especially the PCu/PCC and the left angular gyrus/inferior parietal lobule, may provide a neurological explanation for the well-accepted cognitive model of PD, in which PD patients often focus on internal and somatosensory stimuli, interpret normal bodily sensations as signs of disaster, and identify the safe stimuli and the environment as a threat, then causing a fear response.[46,47] Although almost all of the amygdala subregions showed similar increased functional connectivity to the whole amygdala with PCu/PCC, we found a distinct change of the rsFC between the lBLA and the left angular gyrus/inferior parietal lobule, which increased in PD group. To some extent, the results were consistent with our hypothesis that specific functional connectivity abnormality occurs in the amygdala subregions. In addition, we found that the more anxious the PD patients were, the stronger the connectivity between the lSFA and the left PCu, which provided further evidence that the connectivity between the amygdala subregions and the DMN plays an important role in the pathophysiological basis of PD. It provides a basis for the further investigation about the hyper-connectivities between amygdala subregions and DMN in PD. There are some limitations in our study. First, the nature of this cross-sectional study may obscure some dynamic changes over the course of the disease. Longitudinal studies across acute and remission periods might provide new insights into the characteristics of the alterations of functional connectivity, and determine whether these changes are traits or state markers in PD. Second, as the serious PD patients often fail to complete the fMRI scan, the PD patients in our study were mostly mild or moderate, and the distribution of PDSS-CV score in our study was narrow, which may affect the statistical analysis results. Moreover, we selected the seed regions based on template,[27] and this strategy makes our analysis easier but ignores the individualization of the amygdala and its subregions.[48] A precise understanding of specificity of abnormal functional connectivity of the amygdala subregions is needed in the future by using individualized parcellation of amygdala and its subregions. In conclusion, PD patients showed consistent hyper-connectivity in the amygdala and its subregions (except the rSFA) with PCu/PCC. Besides, this research found differential changes in function connectivity among amygdala subregions–an increased rsFC between the lBLA and the left angular gyrus/inferior parietal lobule. Moreover, most of the brain regions with altered rsFC were located in the DMN, which indicates that the rsFCs between the DMN and the amygdala as well as its subregions play an important role in the pathogenesis of PD. Future studies can further explore the possibility to take the increased rsFC between the amygdala and the DMN as a biomarker for the diagnosis of PD. The authors are grateful to the participants and their families for contributing to this research. The author would also like to thank Daning Chen, Hang Wu, Xiongying Chen, and Zhifang Zhang for their assistance in recruitment of participants and data analysis. This work was funded by the Capital's Funds for Health Improvement and Research (No. 2020-1-2121), the Beijing Municipal Science and Technology Commission (No. Z181100001718077), and Special Funding of the Beijing Municipal Administration of Hospitals Clinical Medicine Development (No. ZYLX201815). None. How to cite this article: Zhang P, Yang X, Wang Y, Liu H, Meng L, Yan Z, Zhou Y, Li Z. Increased functional connectivity of amygdala subregions in patients with drug-naïve panic disorder and without comorbidities. Chin Med J 2023;136:1331–1338. doi: 10.1097/CM9.0000000000002439 Supplemental digital content is available for this article.

5 6 2023

Chinese Medical Journal

10.1097/CM9.0000000000002439

Molecular Mechanisms in the Genesis of Seizures and Epilepsy Associated With Viral Infection

Seizures are a common presenting symptom during viral infections of the central nervous system (CNS) and can occur during the initial phase of infection (“early” or acute symptomatic seizures), after recovery (“late” or spontaneous seizures, indicating the development of acquired epilepsy), or both. The development of acute and delayed seizures may have shared as well as unique pathogenic mechanisms and prognostic implications. Based on an extensive review of the literature, we present an overview of viruses that are associated with early and late seizures in humans. We then describe potential pathophysiologic mechanisms underlying ictogenesis and epileptogenesis, including routes of neuroinvasion, viral control and clearance, systemic inflammation, alterations of the blood-brain barrier, neuroinflammation, and inflammation-induced molecular reorganization of synapses and neural circuits. We provide clinical and animal model findings to highlight commonalities and differences in these processes across various neurotropic or neuropathogenic viruses, including herpesviruses, SARS-CoV-2, flaviviruses, and picornaviruses. In addition, we extensively review the literature regarding Theiler’s murine encephalomyelitis virus (TMEV). This picornavirus, although not pathogenic for humans, is possibly the best-characterized model for understanding the molecular mechanisms that drive seizures, epilepsy, and hippocampal damage during viral infection. An enhanced understanding of these mechanisms derived from the TMEV model may lead to novel therapeutic interventions that interfere with ictogenesis and epileptogenesis, even within non-infectious contexts.

Edited by: David Blum, INSERM U1172 Centre de Recherche Jean Pierre Aubert, France Reviewed by: Maximilian Lenz, University of Freiburg, Germany; Madhuvika Murugan, New Jersey Institute of Technology, United States This article was submitted to Brain Disease Mechanisms, a section of the journal Frontiers in Molecular Neuroscience Seizures are a common presenting symptom during viral infections of the central nervous system (CNS) and can occur during the initial phase of infection (“early” or acute symptomatic seizures), after recovery (“late” or spontaneous seizures, indicating the development of acquired epilepsy), or both. The development of acute and delayed seizures may have shared as well as unique pathogenic mechanisms and prognostic implications. Based on an extensive review of the literature, we present an overview of viruses that are associated with early and late seizures in humans. We then describe potential pathophysiologic mechanisms underlying ictogenesis and epileptogenesis, including routes of neuroinvasion, viral control and clearance, systemic inflammation, alterations of the blood-brain barrier, neuroinflammation, and inflammation-induced molecular reorganization of synapses and neural circuits. We provide clinical and animal model findings to highlight commonalities and differences in these processes across various neurotropic or neuropathogenic viruses, including herpesviruses, SARS-CoV-2, flaviviruses, and picornaviruses. In addition, we extensively review the literature regarding Theiler’s murine encephalomyelitis virus (TMEV). This picornavirus, although not pathogenic for humans, is possibly the best-characterized model for understanding the molecular mechanisms that drive seizures, epilepsy, and hippocampal damage during viral infection. An enhanced understanding of these mechanisms derived from the TMEV model may lead to novel therapeutic interventions that interfere with ictogenesis and epileptogenesis, even within non-infectious contexts. Seizures are common presenting symptoms of viral infections of the central nervous system (CNS), and can occur during the acute phase of infection (“early” or acute symptomatic seizures or status epilepticus), after recovery (“late” or spontaneous seizures; indicating the development of acquired epilepsy), or both (Misra et al., 2008; Vezzani et al., 2016). These two types of epileptic seizures have different underlying mechanisms and prognostic implications (Löscher et al., 2015). Over 100 different neurotropic viruses cause encephalitis (i.e., inflammation of the brain parenchyma) in humans, and of these, several play a significant role in the development of seizures and epilepsy (Table 1). Some types of viral encephalitis occur sporadically in worldwide distribution, while others have restricted geographic ranges, often related to specific viral vectors and hosts (Theodore, 2014). The incidence both of acute symptomatic seizures and subsequent epilepsy varies with the specific type of viral encephalitis (mainly dependent on the affected brain regions), the patient’s age, delays in starting treatment, and possibly the degree of cortical inflammation (Misra et al., 2008; Michael and Solomon, 2012; Theodore, 2014). In contrast to encephalitis (or encephalomyelitis), viral infection confined to the meninges rarely causes seizures and does not increase the risk for later epilepsy (Theodore, 2014). Common viruses associated with seizures and epilepsy in humans. The data shown are based on extensive literature research, using Pubmed and Google Scholar. Evidence of seizures or epilepsy is indicated by “+”; preliminary or anecdotal evidence by “(+)”; and lack of published data by “?”. Positive-sense single-stranded RNA viruses are indicated by +ssRNA, negative-sense single-stranded RNA viruses by −ssRNA, and double-stranded DNA viruses by dsDNA. As will be discussed later in this review, the mechanisms by which neurotropic viruses enter the brain may by themselves lead to ictogenic and epileptogenic brain alterations, particularly when the mechanism of invasion involves damaging the blood-brain barrier (BBB). Most acute and persistent viral infections begin in the periphery and only rarely spread into the CNS, because the CNS is protected from most virus infections by effective immune responses and specific barriers, such as the BBB or the blood-cerebrospinal fluid (CSF) barrier (Koyuncu et al., 2013; Löscher and Friedman, 2020). However, neurotropic viruses may enter the brain through multiple routes (Figure 2 and Table 2). Most commonly, they spread hematogenously, i.e., across the BBB, but they can also invade the brain via the olfactory nerves in the nasal mucosa, through the choroid plexus into the CSF, or via trans-synaptic retrograde transport following infection of peripheral nerves (Nath and Johnson, 2021). Mechanisms of neuroinvasion by neurotropic viruses. Note that, at least in part, these data are from preclinical models. For details, see Koyuncu et al. (2013), Cain et al. (2019), and Nath and Johnson (2021). Abbreviations: BCEC, brain capillary endothelial cell; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; HSV, herpes simplex virus; CMV, cytomegalovirus; HHH, human herpesvirus; EPV, Epstein-Barr virus. The BBB is a dynamic, highly selective barrier primarily formed by brain microvascular endothelial cells (BMECs) connected by tight junctions that separate the circulating blood from the brain parenchyma (Löscher and Friedman, 2020). The tight junctions between the BMECs limit the paracellular flux of hydrophilic and macro-molecules as well as the entry of cells across the BBB, while nutrients such as glucose and amino acids enter the brain via specific membrane transporters. As shown in Figure 2, in addition to endothelial cells, the BBB is composed of the capillary basal or basement membrane, pericytes embedded within the basal membrane, and the glia limitans, formed by astrocytic end-feet processes that surround the endothelial cells and add to the barrier properties (Löscher and Friedman, 2020). As summarized in Figure 2 and Table 2, viruses can use diverse routes of neuroinvasion that also dictate which brain regions are affected by the virus. Although the BBB protects the brain from pathogens, viruses can penetrate the barrier by several means. One way is through direct infection of the brain endothelium resulting in transcellular transport into the CNS (Table 2 and Figure 2). Examples of viruses thought to enter the CNS through this route include West Nile virus (WNV) and poliovirus (Coyne et al., 2007; Verma et al., 2009). Pathogens also may cross the BBB paracellularly via disruption of the tight junctions or by damaging BMECs (Cain et al., 2019). Strategies used by neurotropic pathogens in this regard include induced secretion of tight junction-disrupting proteases and toxins, hijacking of host inflammatory and immune responses, and lytic damage of BMECs. Further, it is thought that viruses may enter the brain at regions of heightened permeability (Nath and Johnson, 2021). The BBB is heterogeneous throughout the CNS, and some regions, such as the circumventricular organs, are more permeable than others due to the absence of tight junctions (Löscher and Friedman, 2020). Alternatively, viruses may penetrate the BBB and enter the brain parenchyma through the trafficking of infected leukocytes, often termed “The Trojan Horse” pathway (Outram et al., 1975; Williams and Blakemore, 1990). Phagocytic leukocytes contribute to the clearance of viral, bacterial, and parasitic infections. However, after the internalization of the virus or direct infection of the leukocytes, pathogens may exploit the migratory capabilities of these cells to cross the BBB and lead to CNS infection (Figure 2 and Table 2). Other pathogens, e.g., mumps and rabies viruses, use hematogenous routes to gain access to the CSF compartment (Table 2). Another mechanism of virus invasion into the CNS is via the olfactory system (Figure 2), which provides a unique and directly accessible portal of entry to the CNS from the periphery (Koyuncu et al., 2013). As shown in Table 2, several viruses may infect neurons in the nasal olfactory epithelium. Spread to the CNS occurs via anterograde axonal transport along the olfactory nerve into the brain (Figure 2). The olfactory epithelium is well protected from most common infections by mucus and the presence of several pathogen recognition receptor systems (Kalinke et al., 2011). However, there is evidence that pathogens such as herpes simplex virus type-1 (HSV-1), influenza A virus (IAV), parainfluenza viruses, rabies virus, and, more recently, SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2) can enter the CNS through the olfactory route (Table 2). Following CNS entry via the olfactory system, the virus may spread to other parts of the brain, e.g., using axonal transport via the lateral olfactory tract to the hippocampus, which often acts as a focus in the development of epilepsy and cognitive impairment following virus infections (Vezzani et al., 2016). Viruses such as the herpes viruses and rabies virus infect peripheral neurons (Table 2), leading to anterograde or retrograde transport of virions or viral ribonucleoprotein complexes within axons into the CNS, followed by trans-synaptic transport and infection of new neurons (Vezzani et al., 2016). Another possible mechanism of viral invasion is just the entry of viral proteins and not the entire virus into the CNS. For instance, Rhea et al. (2021) reported that the S1 subunit of the spike protein of SARS-CoV-2 crosses the mouse BBB by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 (ACE2) is involved in brain and lung uptake, but not in kidney, liver or spleen uptake. In a subsequent in vitro study, the S1 protein was shown to cross the human brain endothelial cell barrier effectively (Petrovszki et al., 2022). Central nervous system viral infections are a major cause of death and disability globally (Manglani and McGavern, 2018). The spatial distribution of CNS infection and localization of the consequent immune response results in meningitis (inflammation restricted to the meninges), meningoencephalitis (inflammation of the meninges and brain parenchyma), myelitis (inflammation of the spinal cord), encephalitis (inflammation of the brain parenchyma), or encephalomyelitis (inflammation of the brain and spinal cord). The manifestations of CNS viral infection include fever, altered mental state, neurocognitive impairment, seizures, brain damage, stroke, and death. For many viruses, a robust innate immune response is readily elicited at CNS barriers, including the meninges, the perivascular space, and the ventricular system, which prevents further spread into the subjacent parenchyma (Vincenti and Merkler, 2021). At these CNS barriers, specialized macrophage populations, including dural, leptomeningeal, perivascular, and choroid plexus macrophages, are collectively referred to as CNS-associated macrophages (CAMs) (Kierdorf et al., 2019). Early pathogen detection by CAMs and CNS-resident microglia triggers a disease-associated signature and the release of pro-inflammatory cytokines and chemoattractants (Vincenti and Merkler, 2021). CAMs thereby initiate an inflammatory response that recruits other immune cells, including neutrophils and monocytes. While these innate immune response mechanisms do not directly clear the virus, per se, they are vital for the initiation of cytokine-mediated antiviral programs and the subsequent recruitment of adaptive antiviral T cells. Ultimately, the control and clearance of most CNS viral infections depend on the adaptive immune system, including both newly trained antiviral cytotoxic T cells and re-expanded populations of memory lymphocyte subsets (Libbey and Fujinami, 2014). The latter surveil the CNS to rapidly detect invading or re-activating viruses and provide immediate responses toward previously encountered antigens (Vincenti and Merkler, 2021). If a virus invades the CNS as described above, innate immune responses are mainly coordinated by microglia, i.e., the resident macrophages and primary innate immune cells of the CNS (Chen et al., 2019), and by astrocytes (Klein et al., 2019). Indeed, once thought to be immune-privileged, the CNS is now known to be immune-competent, dynamic, and in direct contact with the peripheral immune system (Manglani and McGavern, 2018). However, the specific role of microglia and other CNS resident cells in this process and their interactions with CNS infiltrating immune cells, such as blood-borne monocytes and T cells, are only incompletely understood. At least in part, this is due to the problems of differentiating invading monocytes from activated microglia in the brain and the lack of selective tools to manipulate these two types of myeloid cells (Greter et al., 2015; Butovsky and Weiner, 2018; Spiteri et al., 2022). Because of the BBB, peripheral monocytes are not found in the CNS parenchyma unless there is overt damage to the barrier or unless pathogen-induced chemokine responses in the brain parenchyma are sufficient to drive monocyte infiltration across the barrier. Iba-1 (ionized calcium-binding adaptor molecule-1) is widely employed as an immunohistochemical marker for both ramified and activated microglia; however, Iba-1 does not discriminate between microglia and peripheral monocytes that have infiltrated the brain (Jeong et al., 2013). Flow cytometry using the expression of cell surface markers such as CD45 and CD11b is widely used to differentiate microglia from CNS invading monocytes (Prinz et al., 2011; Butovsky and Weiner, 2018); however, during neuroinflammation microglia upregulate CD45 expression and may therefore become indistinguishable from monocytes (Yamasaki et al., 2014; Greter et al., 2015; Käufer et al., 2018a). Recent evidence suggests that surface expression of Ly6C/G molecules may adequately distinguish monocytes from microglia (Howe et al., 2022), though as monocytes differentiate into tissue macrophages they likely become, once again, indistinguishable from resident microglia. Adaptive inflammation-associated changes may also affect the specificity of more recent microglia markers such as TMEM119, further blurring the distinction between microglia and infiltrating monocytes (Bennett et al., 2016; Butovsky and Weiner, 2018). Finally, recent single-cell analyses have shown that microglia exhibit a much higher spatial, temporal, and functional diversity than previously thought (Masuda et al., 2020; Sankowski et al., 2021). In several viral brain infections, activated microglia appear to be involved in both the inhibition of viral replication and in the induction of neurotoxicity, indicating the dual nature of microglia: they contribute to the defense of the CNS but also bear responsibility for CNS damage (Rock et al., 2004; Chhatbar and Prinz, 2021; Figure 3). Microglial phenotypes were, in the past, characterized by the presence of particular cell surface molecules and the expression of specific sets of cytokines and were classified as either M1-like (exhibiting pro-inflammatory signaling and neurotoxicity) or M2-like (participating in the resolution of inflammation) (Butovsky and Weiner, 2018). However, with the help of newly developed technologies, including single-cell RNA-sequencing, quantitative proteomics, and epigenetic studies, it is now clear that this simplistic view of microglial phenotypes does not adequately describe the complex physiology and pathophysiology of microglial cells (Masuda et al., 2020; Sankowski et al., 2021; Waltl and Kalinke, 2022). Microglia expresses various pattern recognition receptors (PRRs) that recognize viral signatures called pathogen-associated molecular patterns (PAMPs) (Bachiller et al., 2018; Gern et al., 2021). Upon stimulation by PAMPs, microglia release several pro- and anti-inflammatory cytokines such as monocyte chemoattractant protein 1 (MCP1 aka CCL2), interleukin (IL)-1β, type I interferon (IFN), IFNγ, and tumor necrosis factor-α (TNF-α) (O’Shea et al., 2013). This microglial response likely recruits inflammatory monocytes during the acute phase and contributes to CNS recruitment of antiviral CD8+ T cells throughout infection. Recruitment of both innate and adaptive immune cells is necessary for effective control of infection, with the innate response limiting viral replication and the adaptive response clearing the virus via both cytolytic and non-cytolytic mechanisms (Griffin, 2010; Libbey and Fujinami, 2014). However, as with the dual role of microglia, infiltrating monocytes contribute to neurotoxicity, synaptic dysregulation, and ictogenesis (Howe et al., 2012a,b; Cusick et al., 2013; Varvel et al., 2016; Cusick et al., 2017; Howe et al., 2017; Käufer et al., 2018a; Figure 3). Recovery from infection requires non-cytolytic clearance of the virus from the CNS to avoid further damage to tissue (Griffin and Metcalf, 2011). B cell production of antiviral antibodies (Bartlett and Griffin, 2020), T cell production of IFN-γ (Milora and Rall, 2019), and other immune responses within the infected nervous system are important for non-cytolytic clearance of infectious virus and viral RNA and also for prevention of viral reactivation and recrudescence (Manglani and McGavern, 2018). Microglia and other neural cells exert direct antiviral effects by producing type I interferons that consequently induce autocrine and paracrine expression of IFN-stimulated genes (ISGs), resulting in viral control and hardening of neural cell susceptibility to further infection (Chen et al., 2019). These signals also induce MHC class I expression and facilitate the presentation of viral peptides that are recognized by antiviral T cells. Infiltrating lymphocytes and natural killer cells, recruited by the same processes that induce type I interferons, produce IFN-γ which drives intracellular processes that block viral replication and enhance the destruction of viral material via autophagic and oxidative mechanisms (Lee and Ashkar, 2018). However, despite this symphony of antiviral responses, some pathogens persist in the CNS (Griffin and Metcalf, 2011; Nath and Johnson, 2021), contributing to ongoing tissue damage and neuroinflammatory processes that exacerbate the consequences of infection. Restricted viral replication within the context of persistent infection in the absence of sterilizing immunity results in chronic neuroinflammation (Nath and Johnson, 2021). Viral mechanisms that contribute to persistence include the route of viral entry into the CNS, viral immune evasion strategies, and viral spread to permissible cells (Nath and Johnson, 2021). In parallel, host genetics contribute significantly to viral clearance versus persistence, as exemplified by TMEV infection in SJL versus B6 mice (Howe et al., 2012b; Gerhauser et al., 2019). Viruses may also enter a latent state within the CNS, marked by the continued presence of viral genomic material but limited gene expression and no replication. A crucial component of such cryptic infection is the reversion to the active expression of the complete viral genome and resurgent production of infectious virions. Herpesviruses such as Epstein-Barr virus (EBV) are canonical latent infectious agents (Speck and Ganem, 2010), and human herpesvirus (HHV)-6, a nearly ubiquitous pathogen in children, establishes latency in the CNS (Dunn et al., 2020). Later reactivation of HHV-6 may drive limbic encephalitis, and, as described below, induce seizures and temporal lobe epilepsy (TLE). Overall, the detection of persistent or latent viruses in the CNS is severely hampered by inaccessibility and the field still has much to learn about the influence of such infections on the development of later-life neurological disorders, ranging from Alzheimer’s disease and multiple sclerosis (MS) to epilepsy. As shown in Figure 4, by definition, “early” or acute symptomatic seizures are seizures that occur during the initial phase (typically the first week) of CNS infection, whereas “late” or unprovoked (spontaneous) recurrent seizures develop in surviving patients after a latent period of weeks, months, or years following the acute phase (Löscher et al., 2015). In more general terms, acute symptomatic seizures occur in a close temporal relationship with the initial infection and typically subside once the acute insult is over, usually without recurrence (Singhi, 2011). Early seizures are not a prerequisite for late seizures but increase the risk of spontaneous, unprovoked seizures (i.e., epilepsy), presumably because early seizures are an indicator of injury that leads to maladaptive changes in neural circuitry (Klein et al., 2018). In addition to the dysregulation of synapses incurred by the electrophysiological influence of an early seizure associated with CNS viral infection, the infection-associated inflammatory response elicited in resident microglia and generated by infiltrating leukocytes also confers maladaptive synaptic changes that lead to persistent hyperexcitability. Such changes include morphological alteration of synaptic spine structure (Tomasoni et al., 2017), alterations in the balance of inhibitory and excitatory neurons and synaptic channels (Habbas et al., 2015), and transcriptional reprogramming that alters neuronal excitability (Buffolo et al., 2021). It is estimated that half of all patients with encephalitis experience acute symptomatic seizures, and approximately 4% develop status epilepticus, a medical emergency in which a patient has a seizure lasting longer than 5 min or has multiple discrete seizures between which consciousness is not fully recovered. An episode of status epilepticus, especially one lasting 30 min or more, greatly increases the risk of developing epilepsy (Barnard and Wirrell, 1999). Epilepsy exists when someone has an unprovoked seizure and their brain “demonstrates a pathologic and enduring tendency to have recurrent seizures” (Fisher et al., 2014). More specifically, in survivors of viral infections, epilepsy is diagnosed when an individual has: (1) at least two unprovoked or reflex seizures > 24 h apart, (2) one unprovoked or reflex seizure and a probability of having another seizure similar to the general recurrence risk after two unprovoked seizures (≥60%) over the next 10 years, or (3) an epilepsy syndrome (Fisher et al., 2014). Importantly, early and late seizures may look very similar, both behaviorally and by EEG (Löscher et al., 2015). Thus, determining whether a patient or group of patients developed epilepsy after viral infection is not trivial, but necessitates a thorough review of symptoms and medical history and detailed diagnostic testing, including high-resolution EEG, to adequately diagnose epilepsy and determine the cause of seizures. This explains why it is often not yet clear, particularly for infections occurring in the developing world, whether a virus infection causes epilepsy or only early seizures. For the current review, we performed an extensive literature search, using Pubmed and Google Scholar, to find studies that unequivocally identified epilepsy as an outcome in patients infected with a variety of neurotropic and neuropathogenic viruses. The outcome of this search is shown in Table 1, demonstrating that many more viruses than thought before can lead to unprovoked seizures and epilepsy. As shown in Table 1, a variety of different RNA and DNA viruses have been reported to cause acute symptomatic seizures and subsequent epilepsy. Among the viruses shown in Table 1, the high prevalence and spread of arthropod-borne viruses (arboviruses) make them an important cause of viral encephalitis and associated seizures in humans, with between 10 and 35% of patients infected with these viruses displaying some form of seizure (Getts et al., 2008; Singhi, 2011; Zheng et al., 2020). Among the various arboviruses, flaviviridae such as WNV, Japanese encephalitis virus (JEV), Zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) have been reported to induce both early and, in survivors, late (spontaneous) seizures (Table 1). JEV is the single largest cause of acute epidemic encephalitis worldwide (Singhi, 2011). Acute symptomatic seizures are reported in 50–80% of cases and are much more frequent in children than in adults. The seizures are generalized or focal with secondary generalization, single or multiple, and may present as status epilepticus. Late-onset epilepsy is less common in JEV (Singhi, 2011; Chen et al., 2021). Concerning congenital ZIKV syndrome, recent reports show that epileptic seizures are among the main neurological outcomes of this syndrome (Table 1). Among the sporadic viral encephalitides, herpes simplex encephalitis (HSE) is perhaps most frequently associated with epilepsy, which may often be severe (Misra et al., 2008). Seizures may be the presenting feature in 40–70% of patients during acute infection and the frequency of epilepsy in survivors may be 40–60% (Theodore, 2014). The propensity to cause seizures is probably related to viral spread along olfactory pathways to limbic structures including the temporal lobe, insula, and cingulate cortex. Other potentially neurotropic viruses, such as measles, varicella, mumps, IAV, and enteroviruses may cause seizures depending on the area of the brain involved (Misra et al., 2008). COVID-19 (coronavirus disease 2019), the global pandemic caused by SARS-CoV-2, is considered to be primarily a respiratory disease, but SARS-CoV-2 infection affects multiple organ systems including the CNS (Mishra and Banerjea, 2020). Numerous reports have described seizures in people with COVID-19 (Asadi-Pooya, 2020; Asadi-Pooya et al., 2021; Doyle, 2021; Nolen et al., 2022), though it is unclear how many of these seizures arise as a complication of systemic inflammation, peripheral organ damage, and vascular injury versus more direct infection-related effects on the CNS. It is also too early to determine whether COVID-19 is associated with epilepsy, although several anecdotal reports suggest de novo epilepsy in these patients (Elgamasy et al., 2020; Nikbakht et al., 2020). In children, seizures may be the main presenting manifestation of acute SARS-CoV-2 infection (Kurd et al., 2021). In the as-yet largest study on neurological manifestations of COVID-19, seizures were observed in 74 of 4491 patients (1.6%), which was the third most common neurological manifestation after encephalopathy and stroke (Frontera et al., 2021). No patient had meningitis/encephalitis or myelopathy/myelitis that was conclusively related to direct SARS-CoV-2 invasion of the CNS. However, these findings do not eliminate the possibility of direct CNS invasion of SARS-CoV-2. Indeed, more recently, olfactory transmucosal SARS-CoV-2 invasion has been described as a port of CNS entry in individuals with COVID-19 (Meinhardt et al., 2021). Some viruses, including HHV-6, IAV, adenovirus, and rhinovirus, are associated with febrile seizures, i.e., seizures that are triggered by fever, typically above 38.3°C. These seizures are the most common type of convulsions in infants and young children (Millichap and Millichap, 2006; Epstein et al., 2012; Rudolph et al., 2021). Most febrile seizures last only a few minutes and are not associated with an increased risk of later spontaneous seizures. However, multiple or prolonged febrile seizures, including febrile status epilepticus (fSE), are a risk factor for epilepsy (Shinnar, 2003). Of greatest concern is the small group of children with febrile seizures lasting longer than 30 min. In these children, the risk of developing epilepsy is as high as 30–40%, though the condition may not develop until many years later. The prospective FEBSTAT study examines the consequences of fSE and is clarifying the relationship between fSE, hippocampal atrophy, hippocampal sclerosis, and the development of subsequent TLE and cognitive impairment (Hesdorffer et al., 2012). As such, this study will be instrumental in determining the role of structural hippocampal alterations as a potential mechanism of TLE. Recent data from the FEBSTAT study suggest that prolonged febrile seizures injure the hippocampus (Shinnar et al., 2012; Lewis et al., 2014; McClelland et al., 2016). Febrile infection-related epilepsy syndrome (FIRES), a subtype of new-onset refractory status epilepticus (NORSE), is a catastrophic epileptic syndrome that strikes previously healthy children between the age of 2 and early adulthood and has unknown pathogenesis and few treatments (Fox et al., 2017; Sculier et al., 2021; Lattanzi et al., 2022; Nausch et al., 2022). Affected children experience a non-specific illness with fever starting between 2 weeks and 24 h before the onset of prolonged refractory status epilepticus. In a few cases, specific pathogens, including rhinovirus, respiratory syncytial virus, and EBV, were identified in serum or nasopharyngeal aspirates (Venkatesan and Benavides, 2015). However, despite extensive testing, pathogens have not been identified in the CNS, suggesting that a systemic infection induces the CNS dysfunction, potentially by triggering inflammation that is communicated across the BBB, inducing sterile encephalitis (Ravizza et al., 2018; Vezzani et al., 2019). The outcome of FIRES varies with the length of the acute phase and is usually poor, with up to 30% of cases ending in death and 60–100% of survivors developing permanent intellectual disability and drug-resistant epilepsy (Fox et al., 2017; Tan et al., 2021). The occurrence of febrile seizures and FIRES, as well as the occurrence of seizures in COVID-19 patients, suggests that systemic inflammatory responses to viral infection in the absence of neuroinvasion and true encephalitis may be an important pathogenic mechanism in driving seizures and epilepsy. Fever and high levels of circulating inflammatory cytokines alter BBB permeability (Danielski et al., 2018; Remsik et al., 2021) and may permit the transmission of inflammation into the CNS. These events may also facilitate viral entry into the CNS that otherwise would not occur, resulting in transient neural infection or PAMP-induced PRR signaling that drives microglial activation in the absence of leukocyte infiltration. These mechanisms of infection-associated ictogenesis may explain how viruses that show weak or no neurotropic potential still elicit early seizures that confer heightened risk for later development of epilepsy. Indeed, systemic virus infection-associated indirect neuroinflammation and ictogenesis may be the parallel of sepsis-associated encephalopathy (Gao and Hernandes, 2021). In addition to viral infections as a trigger for ictogenesis and epileptogenesis, such infections may affect disease progression in patients with existing epilepsy (Vezzani et al., 2016; Tan et al., 2021). In particular, the inflammation associated with viral infections contributes to the progression of the disease (see below). The various molecular, structural, and functional alterations in the CNS that are potentially involved in the generation of seizures and epilepsy associated with viral infections are illustrated in Figures 1, 3–5. The mechanisms underlying the generation of early and late seizures vary with the type and location of infection. In general, early seizures are an acute consequence of virus infection, either directly via neuroinvasion and encephalitis or indirectly via systemic inflammation and neuroinflammation. In contrast, late seizures arise from the functional and structural alterations that drive epileptogenesis, a multifactorial process that is outlined in Figure 4. Interactions of viruses and the central nervous system. Modified from Vezzani et al. (2016). Routes of virus invasion into the brain. In addition to the routes illustrated in the figure, viruses may enter the central nervous system (CNS) via the choroid plexus, i.e., the blood-CSF barrier (see Table 2). Modified from Löscher and Potschka (2005), Löscher and Friedman (2020), and Sulzer et al. (2020). The acute inflammatory and neuroinflammatory responses to CNS viral infection exert both protective and injurious effects. Leukocyte infiltration in response to chemokine production induced by pattern recognition receptor binding to viral components and endogenous alarmins results in potentially pathogenic alterations at the blood-brain barrier and leads to a robust release of cytokines that are critical for enhancing viral control before the development of an adaptive antiviral response. However, these cytokines also disrupt synaptic function and homeostasis, leading to neuronal injury and changes in excitability that confer a pro-ictogenic effect. Ultimately, viral control and clearance from the CNS is a trade-off between exuberant innate immune responses and consequent cellular and circuit damage. Steps in the development and progression of acquired epilepsy (often temporal lobe epilepsy) as a consequence of viral infections. The term epileptogenesis includes processes that take place before the first spontaneous seizure occurs, which render the brain susceptible to spontaneous recurrent seizures and processes that intensify seizures and make them more refractory to therapy (progression). The concept illustrated in the figure is based on both experimental and clinical data. Adapted from Löscher et al. (2008). Pathophysiological cascade of events leading from viral infection to inflammation to seizures and epilepsy. See text for details. Modified from Vezzani et al. (2011) and Vezzani et al. (2019). Hematogenous transmission of virus to the CNS involving either BMEC infection, damage to the tight junctions, or both, results in changes to BBB integrity that are likely an essential mechanism of subsequent ictogenesis and epileptogenesis (Löscher and Friedman, 2020). One hallmark of a damaged BBB is the extravasation of albumin from the blood to the brain parenchyma (Friedman et al., 2009). In the brain parenchyma, albumin can be taken up or bound to neurons, astrocytes, and microglial cells. In astrocytes, albumin can be taken up via transforming growth factor-beta (TGF-β) receptors. This is followed by downregulation of inward rectifying potassium channels (Kir 4.1), water channels (aquaporin 4; AQP4), and glutamate transporters in these astrocytes (Löscher and Friedman, 2020). As a result, the buffering of extracellular potassium and glutamate is reduced, which facilitates N-methyl-D-aspartate (NMDA) receptor-mediated neuronal hyperexcitability and eventually induces epileptiform activity (Löscher and Friedman, 2020). TGF-β receptor signaling is further associated with transcriptional changes involved in inflammation, alterations in extracellular matrix (specifically the perineuronal nets around inhibitory interneurons), excitatory synaptogenesis, and pathological plasticity, all considered important mechanisms that contribute to lowering the seizure threshold during epileptogenesis (Löscher and Friedman, 2020). As a proof-of-concept that albumin extravasation plays a crucial role in the generation of seizures, the angiotensin II type 1 (AT1) receptor antagonist, losartan, which blocks brain TGF-β receptor signaling, was shown to prevent epilepsy in different models of epileptogenesis (Swissa et al., 2019). For many decades, the limbic system in the temporal lobes, including the hippocampal formation and parahippocampal areas such as the piriform, perirhinal, and entorhinal cortices, have been known to play a crucial role in the development of seizures and epilepsy (Walter, 1969; Meldrum, 1975; Ribak et al., 1992; Engel, 1996; Löscher and Ebert, 1996; Chatzikonstantinou, 2014; Scharfman, 2019). The hippocampus is considered by many to be the generator of TLE, the most common type of epilepsy in adults and a frequent consequence of viral infections (Vezzani et al., 2016). TLE is typically associated with hippocampal sclerosis, a neuropathological condition with severe neuronal cell loss and gliosis in the hippocampus, specifically in the CA1 (Cornu Ammonis area 1) region and subiculum of the hippocampus proper and in the hilus of the dentate gyrus (Blümcke et al., 2002). Hippocampal sclerosis was first described in 1880 by Wilhelm Sommer as an etiological component of epilepsy (Sommer, 1880). In addition to neuron loss, aberrant sprouting of dentate granule cell mossy fibers in mesial TLE is thought to underlie the creation of aberrant circuitry that promotes the generation or spread of spontaneous seizure activity (Sutula and Dudek, 2007; Scharfman, 2019). Surgical removal of the sclerotic hippocampus in drug-resistant patients often improves or even cures TLE (Löscher et al., 2020). Thus, these structural changes in the hippocampal formation provide a mechanism by which viral infections could induce seizures and epilepsy. As discussed above, some viruses may be more epileptogenic due to their anatomic distribution, as in the case of HSV, with a propensity to affect the temporal lobes, including the hippocampus (Theodore, 2014). HSV causes widespread inflammation, edema, and parenchymal necrosis (Theodore, 2014). Experimental corneal inoculation of HSV-1 in BALB/c mice led to increased CA3 pyramidal cell excitability and aberrant mossy fiber sprouting in the hippocampus as well as clinical seizures (Wu et al., 2003). Remarkably, after initial infection, HSV can establish persistent latent infections in the CNS, acting as a continuous source of HSE recurrence (Zhang et al., 2020). Concerning the neurotropic virus HHV-6, several studies and a recent meta-analysis suggest a pathogenic role of HHV-6B infection in the development of mesial TLE, especially when associated with hippocampal sclerosis and a history of febrile seizures (Wipfler et al., 2018; Bartolini et al., 2019; Wang and Li, 2021). HHV-6, which is ubiquitous and infects most people when they are children, establishes latent infections in the CNS, especially in the hippocampus and amygdala, and is associated with neurologic diseases, including TLE (Wang and Li, 2021). In a meta-analysis of studies that detected HHV-6 genomic DNA or protein in brain samples from the hippocampus of people with mesial TLE, HHV-6 DNA was detected in 19.6% of all TLE patients compared to 10.3% of all controls (P < 0.05) (Wipfler et al., 2018). Transcriptional analysis of the amygdala in patients with hippocampal sclerosis revealed higher expression of CCL2 and glial fibrillary acidic protein (GFAP) in HHV-6 positive samples and a positive correlation between viral load and protein expression (Kawamura et al., 2015). As described above, CCL2 is a chemokine that participates in the migration and CNS infiltration of monocytes, in which HHV-6 can establish latent infection (Bartolini et al., 2019). Overexpression of GFAP and CCL2 is associated with neuronal loss and gliosis and has been previously described in resected epileptogenic tissue from the hippocampus (Xu et al., 2011). However, the casual relationship and possible pathological role of HHV-6 in TLE are yet to be elucidated. Infections with ZIKV have also been reported to cause alterations in temporal lobe structures such as the hippocampus, leading to memory and behavioral deficits and seizures (Stanelle-Bertram et al., 2018; Büttner et al., 2019; Raper et al., 2020). This will be discussed in more detail below. Upon viral invasion of the CNS, activation of the innate and adaptive immune response is critical to control viral replication and spread (Libbey and Fujinami, 2014; Figure 3). However, an exuberant innate response to the infection may cause considerable acute bystander pathology, while failing to adequately control viral replication which may lead to persistent smoldering inflammation that results in chronic neuropathology (DePaula-Silva et al., 2021; Figure 3). In general, as illustrated in Figure 5, inflammation plays a prominent role in the mechanisms underlying increased neuronal excitability in both early and late seizures associated with virus infection (Vezzani et al., 2016). Furthermore, oxidative stress is thought to contribute to these processes (Figure 5). As shown in Figures 1, 3, 5, initiation of neuroinflammation may either be the result of neuroinvasion, host danger signal response mediated effects or both. As described above, encephalitis is defined as inflammation of parenchymal CNS tissue that occurs in response to viral replication (Vezzani et al., 2016). Once a virus enters the brain parenchyma, inflammation may result from two mechanisms that are not mutually exclusive. First, viruses may directly infect neurons leading to unconstrained neuronal lysis and death and the release of proinflammatory cytokines and cellular products that act as endogenous danger signals (such as ATP or mitochondria-derived DNA N-formyl peptides) (Vezzani et al., 2016; Di Virgilio et al., 2020; Das et al., 2021). Second, viral PAMPs may activate PRRs on microglia and astrocytes, leading to cytokine and chemokine production that recruits innate immune effectors that drive immunopathology. These inflammatory responses drive acute injury but are also associated with the formation of a residual pathological state marked by continued BBB dysfunction and injury, neuronal death, and persistent neuronal hyperexcitability, all of which may contribute to ictogenesis and epileptogenesis. Viruses may also trigger post-infectious encephalitis or encephalomyelitis, even in the absence of neuroinvasion during the initial infection. Such delayed responses are elicited following the development of T cell- and/or antibody-mediated recognition of self epitopes (Vezzani et al., 2016; Popkirov et al., 2017; Joubert and Dalmau, 2019). Molecular mimicry, epitope spreading, and unmasking of autoreactive lymphocytes (see Figure 1) are the primary mechanisms by which infectious agents induce autoimmunity (Powell and Black, 2001; Cusick et al., 2012; Pape et al., 2019; Gupta and Weaver, 2021). During the acute response to CNS infection, brain resident cells recruit peripheral immune cells to sites of viral infection (Manglani and McGavern, 2018). Among the acute responders, CNS infiltration of monocytes and neutrophils is a hallmark of CNS inflammation, including viral infection (Terry et al., 2012). These cells engage in several potent effector functions including the production and secretion of numerous pro-inflammatory mediators and reactive oxygen species that drive tissue damage (Terry et al., 2012). Monocytes that migrate into the infected brain also differentiate into macrophages, dendritic cells, and, arguably, microglial populations (see below). In addition to invasion of blood-borne immune cells such as monocytes and neutrophils, brain resident innate immune cells, including microglia and astrocytes, also produce proinflammatory cytokines and reactive oxygen species that contribute to inflammation and CNS injury (Figure 5). It has been proposed that the IL-1 cytokine system may play a pivotal role in the development of fSE and mesial TLE (Dube et al., 2005; Dube et al., 2010). IL-1β is the primary cytokine responsible for mediating febrile responses in humans and it is a powerful proconvulsant implicated in ictogenesis and epileptogenesis (Dube et al., 2010; Vezzani et al., 2016; Vezzani et al., 2019). At least in part, this effect of IL-1 β is related to its suppressive action on inhibitory GABA currents and enhancement of NMDA-mediated neuronal Ca2+ influx, resulting in increased glutamatergic excitation (Huang et al., 2011; Mishra et al., 2012; Vezzani and Viviani, 2015). The effects of IL-1β are mediated via IL-1 receptor type 1 (IL-1R1), which is enriched in cortical and hippocampal neurons where it co-localizes and physically associates with the NR2B (GluN2B) subunit of the NMDA receptor (Vezzani and Viviani, 2015). IL-1R1 is activated by IL-1β that is released from neurons, glia, brain endothelial cells, and infiltrating monocytes following inflammasome activation (Labzin et al., 2016; Vezzani et al., 2019). Elevation of IL-1β induces robust release of other proinflammatory cytokines, including IL-6 and CXCL8 (Heida and Pittman, 2005; Vezzani et al., 2016). A recent study that examined the association between plasma cytokines and fSE in children, as well as their potential as biomarkers of acute hippocampal injury, found that levels of CXCL8 and epidermal growth factor (EGF) were significantly elevated after fSE in comparison to controls (Gallentine et al., 2017). However, individual cytokine levels were not predictive of MRI changes in the hippocampus. The nuclear protein high mobility group box 1 (HMGB1), which is released by neurons and macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli and during unconstrained cell death, is thought to play a critical role as a danger signal in virus infection-induced inflammatory responses in the CNS (Wang et al., 2006; Vezzani et al., 2016; Walker et al., 2022). Furthermore, HMGB1 has been implicated in the generation of seizures and epilepsy (Ravizza et al., 2018). As with IL-1β, TNF-α, and IL-6, HMGB1 has pro-ictogenic properties in animal models and affects neuronal function by inducing rapid post-translational changes in glutamate receptor subunit composition and/or phosphorylation (Vezzani et al., 2016). HMGB1 physiologically interacts with nucleosomes, transcription factors, and histones within the nucleus of nearly every cell type but is rapidly translocated to the cytoplasm and released following brain injury and during seizures (Jiang et al., 2020; Murao et al., 2021). Several viruses that cause encephalitis and seizures, including WNV, SARS, TBEV, and IAV, can induce the release of HMGB1 (Wang et al., 2006; Ding et al., 2021). HMGB1 binds to and activates the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and TLR2 (Jiang et al., 2020), inducing signal transduction cascades that drive inflammation. Indeed, activation of IL-1R1 and HMGB1 receptors expressed by microglia and astrocytes orchestrates inflammatory events that result in the release of cytokines and chemokines, induction of the prostaglandin-synthesizing enzyme cyclooxygenase 2 (COX-2), and activation of the complement system, and may thereby subsequently lead to recruitment of leukocytes to the brain (Vezzani et al., 2016). Whereas the processes illustrated in Figure 5 and discussed above would be relevant for all viruses that cause encephalitis and/or sterile inflammation, there are also neuropathophysiological processes and outcomes specific to individual viruses. For instance, the transactivator of transcription (Tat) protein is a major viral protein in HIV that can directly drive neurotoxicity (Atluri et al., 2015). Tat is vital for HIV replication and influences transcription initiation and elongation at the HIV promoter. In addition, however, Tat injures neurons via several different mechanisms, including induction of inflammatory cytokines, impairment of mitochondrial function, and activation of ionotropic glutamate receptors (Atluri et al., 2015). Indeed, Haughey et al. (2001) reported that HIV-1 Tat potentiates the excitotoxicity of glutamate by phosphorylating NMDA receptors, a process that is critically involved in neuronal hyperexcitability, seizures, and epileptogenesis (Ghasemi and Schachter, 2011; Hanada, 2020). The effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a transgenic mouse model constitutively expressing the HIV-1 Tat gene (Zucchini et al., 2013). Stimulus-evoked glutamate exocytosis in the hippocampus and cortex of these mice was significantly increased and was associated with increased seizure susceptibility. In addition to the effects associated with the Tat protein, the HIV type 1 envelope glycoprotein gp120 activates macrophages, which release neurotoxins that affect the glutamate system, leading to activation of voltage-dependent calcium channels and modulation of NMDA signals (Potter et al., 2013). Concerning the SARS-CoV-2 virus, the specific mechanisms by which this virus affects the CNS remain unclear (Pröbstel and Schirmer, 2021). As described above, infection with SARS-CoV-2 may result in psychiatric and neurological symptoms, including seizures; more than 35% of COVID-19 patients develop such symptoms, particularly during severe manifestation of the disease (Tavkar et al., 2021). It is well accepted that the entry of SARS-CoV-2 into a host cell is mediated by ACE2, which functions as an entry receptor (Hoffmann et al., 2020). Membrane-bound ACE2 is a zinc-containing metalloenzyme located on the surface of cells. SARS-CoV-2 downregulates ACE2, with a consequent loss of its catalytic activity (Pacheco-Herrero et al., 2021). Inflammation and thrombosis have been associated with enhanced and unimpeded angiotensin II effects through the ACE2-AT1 receptor axis (Pacheco-Herrero et al., 2021). In the CNS, ACE2 is expressed in the majority of brain regions (e.g., the amygdala, cortex, frontal cortex, substantia nigra, and hippocampus) but mostly at low levels (Chen et al., 2020). Analysis of human and mouse brains showed that ACE2 is expressed predominantly in neurons but also in non-neuronal cells, including astrocytes, oligodendrocytes, endothelial cells, and pericytes (Tavkar et al., 2021). The expression of ACE2 makes CNS cells susceptible to SARS-CoV-2 infection, provided that the virus enters the brain. As summarized in Table 2 and Figure 2, current evidence points to two plausible mechanisms of brain invasion by SARS-CoV-2: (i) entry into the CNS via axonal transport along infected olfactory nerves and then dissemination via trans-synaptic transmission to other brain areas (Montalvan et al., 2020; Yachou et al., 2020; Meinhardt et al., 2021); note that as many as 65% of COVID-19 affected individuals reported hyposmia, anosmia, and ageusia, suggesting the possibility of transsynaptic spread not only via the olfactory route but also along lingual and glossopharyngeal nerves (Figure 2; Sulzer et al., 2020); (ii) entry into the CNS via a hematogenous pathway, either through the infiltration of infected blood cells (usually leukocytes) or through infection of endothelial cells at the BBB. The hematogenous pathway may also involve infection of epithelial cells of the choroid plexus, the building blocks of the blood-CSF barrier (Montalvan et al., 2020; Murta et al., 2020; Vargas et al., 2020; Yachou et al., 2020). Another intriguing mechanism via which SARS-CoV-2 may spread is through the vagus nerve from infected lungs (Jarrahi et al., 2020). Using human brain organoids derived from induced pluripotent stem cells as a valuable tool for investigating SARS-CoV-2 neurotropism, it was found that choroid plexus organoids showed a high rate of infection and supported productive viral replication, consistent with the finding that the choroid plexus exhibits high ACE2 expression (Jacob et al., 2020; Pellegrini et al., 2020). Besides epithelial cells of choroid plexus, neurons, astrocytes, and neural progenitor cells in brain organoids are also susceptible to SARS-CoV-2 infection, although the infection rates for these cell types remain under debate (Tavkar et al., 2021). Overall, replication of SARS-CoV-2 in the CNS remains a controversial issue. Concerning the mechanisms of neurological symptoms such as seizures, many groups argue that the devastating neurological damage caused by SARS-CoV-2 is not a consequence of direct infection of neural cells but rather a result of the severe peripheral hyper-inflammation associated with COVID-19 (Pacheco-Herrero et al., 2021; Tavkar et al., 2021). Among the various consequences of such inflammation, impairment of BBB may be involved in CNS symptoms, as discussed above and illustrated in Figures 3, 5. Furthermore, it has been suggested that endothelial dysfunction in several organs, including the CNS, may be triggered by the interaction between SARS-CoV-2 and ACE2 receptors that are expressed by endothelial cells (Pacheco-Herrero et al., 2021). In patients with COVID-19, magnetic resonance imaging (MRI) detected lesions that are compatible with a cerebral small-vessel disease and with disruption of the BBB (Nassir et al., 2021). More recently, Wenzel et al. (2021) reported structural changes in cerebral small vessels of patients with COVID-19 and elucidated potential mechanisms underlying the vascular pathology. Both in patients and two animal models of SARS-CoV-2 infection, an increase in string vessels was observed in the brain. These structures represent the endothelial cell-free remnants of lost capillaries. Furthermore, the authors also found evidence that BMECs are infected and that the death of BMECs in COVID-19 is secondary to SARS-CoV-2 infection. The SARS-CoV-2 genome encodes two viral proteases that are responsible for processing viral polyproteins into the individual components of the replication and transcription complexes. Wenzel et al. (2021) found that one of them, SARS-CoV-2 Mpro, cleaves the host protein nuclear factor (NF)-κB essential modulator (NEMO), which is known to modulate cell survival and prevent apoptosis and necroptosis. However, other findings suggest that SARS-CoV-2-related neurological complications may be a direct result of the neurovirulent properties of the virus (Shehata et al., 2021). Overall, it has been postulated that there are several different mechanisms involved in COVID-19–associated CNS dysfunction, including activation of inflammatory and thrombotic pathways and, in a few patients, a direct viral effect on the brain endothelium and the brain parenchyma (Bodro et al., 2021). However, further studies are needed to clarify the relative contribution of each of these mechanisms. A recent landmark study used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain (Song et al., 2021). In the first, transgenic mice overexpressing human ACE2 were found to support SARS-CoV-2 neuroinvasion. After intranasal administration, the virus was widely present in neural cells throughout the forebrain. In the second, using human brain organoids, clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons was found. In this study, neuronal infection could be prevented by blocking ACE2. Finally, in autopsies from patients who died of COVID-19, SARS-CoV-2 was detected in cortical neurons. Remarkably, none of the regions of positive viral staining showed lymphocyte or leukocyte infiltration, indicating that SARS-CoV-2 did not invoke an immune response typical of other neurotropic viruses. These findings provide compelling evidence that the brain is a site for the high replicative potential for SARS-CoV-2 and that neurons can become a target of SARS-CoV-2 infection, with devastating consequences for localized ischemia in the brain and cell death, highlighting SARS-CoV-2 neurotropism. The lipid-binding protein apolipoprotein E (ApoE) is the most abundant apolipoprotein in the brain (Flowers and Rebeck, 2020). It is produced predominantly by astrocytes and to some extent microglia. In addition, neurons upregulate ApoE expression in response to excitotoxic injury (Liao et al., 2017). As a major component of very low-density lipoproteins in the brain, ApoE facilitates the transfer of cholesterol and phospholipid between cells. ApoE has been linked with immune responses and neuroinflammation, metabolism, synaptic plasticity, transcriptional regulation, and vascular function by modulating cerebral blood flow, neuronal-vascular coupling, and BBB integrity (Liao et al., 2017). There are three major isoforms (ApoE2, ApoE3, and ApoE4) in humans (Liao et al., 2017). The most common isoform (77–78%) in the general population is E3, whereas E2 is evident in 7–8%, and E4 in 14–16% of individuals (Weisgraber and Mahley, 1996). ApoE4, a strong genetic risk factor for Alzheimer’s disease, is known to lead to BBB dysfunction (Montagne et al., 2020) and has been associated with increased risk for severe COVID-19 (Kuo et al., 2020). Recently, Wang et al. (2021a) tested the neurotropism of SARS-CoV-2 in human induced pluripotent stem cell (hiPSC) models and observed low-grade infection of neurons and astrocytes. They then generated isogenic ApoE3/3 and ApoE4/4 hiPSCs and found an increased rate of SARS-CoV-2 infection in ApoE4/4 neurons and astrocytes. ApoE4 astrocytes exhibited enlarged size and elevated nuclear fragmentation upon SARS-CoV-2 infection. These findings suggest that ApoE4 may play a causal role in COVID-19 severity. Interestingly, ApoE4 has also been associated with seizures. For instance, spontaneous seizures were observed in aged ApoE4 targeted replacement (TR) mice but not in age-matched ApoE2 TR or ApoE3 TR mice (Hunter et al., 2012). In mice with overexpression of ApoE4 (but not ApoE2 or ApoE3), intranasal administration of kainate induced more severe seizures, increased microglial activation, and triggered more hippocampal damage than in wild-type mice (Zhang et al., 2012). In a case-control genetic association study in patients with mesial TLE and hippocampal sclerosis, ApoE4 carriers had an earlier onset of epilepsy than non-carriers (Leal et al., 2017). Thus, in summary, ApoE4 may play a role in seizures observed in viral infections, including COVID-19. As described above, it is a matter of debate whether SARS-COV-2 can enter the brain, but several studies indicate that the SARS-CoV-2 S1 protein can be released from viral membranes, can cross the BBB, and is present in brain cells including neurons (Meinhardt et al., 2021; Rhea et al., 2021; Petrovszki et al., 2022). Thus, Datta et al. (2021) tested the hypothesis that SARS-CoV-2 S1 protein can directly induce neuronal injury. The latter authors found that the S1 protein accumulates in endolysosomes of human cortical neurons and induces aberrant endolysosome morphology and neuritic varicosities, which could contribute to the high incidence of neurological disorders associated with COVID-19. Emerging data suggest that ∼10–40% of patients fail to fully recover after acute COVID-19 infection (Doyle, 2021; Nalbandian et al., 2021). Patients who report symptoms persisting for weeks or months after the acute illness have been termed “long haulers” or described as having “long-COVID.” Long-COVID comprises a variety of symptoms, of which the neurological component prevails, often characterized as post-infectious fatigue syndrome (Sandler et al., 2021). Furthermore, new-onset seizures in people with COVID-19 can potentially extend beyond the acute phase of the infection (Doyle, 2021). The most widely accepted theory on the genesis of these symptoms builds upon the development of microvascular dysfunction similar to that seen in numerous vascular diseases such as diabetes. This can occur through the peripheral activation of ACE2 receptors or through the exacerbating effects of pro-inflammatory cytokines that remain in circulation even after the infection diminishes (Nalbandian et al., 2021). However, at least in part, some of the mechanisms of CNS symptoms discussed above for the acute infection may also play a role in post-COVID symptoms. As discussed above, accumulating evidence suggests a pathogenic role of HHV-6B infection in the development of mesial TLE, and a relationship between viral load and markers that directly (CCL2) or indirectly (GFAP) reflect inflammatory or otherwise injurious processes. How might these observations associating mesial TLE with increased HHV-6 viral detection and increased markers of neuroinflammation and astrocyte activation be mechanistically associated with epilepsy? Inflammation and HHV-6 infection have each been demonstrated to induce dysregulation of glutamate homeostasis in astrocytes, which is hypothesized to play a central role in the pathogenesis of epilepsy (Leibovitch and Jacobson, 2015). In vitro, HHV-6 infection of primary astrocytes has been shown to downregulate levels of glutamate transporter expression, which supports the concomitant observation of decreased glutamate uptake in infected versus uninfected astrocytes (Fotheringham et al., 2008). Inflammatory cytokines, such as IL-1β, can also inhibit the astrocyte reuptake of glutamate (Vezzani and Baram, 2007). Because HHV-6–infected astrocytes have been demonstrated in mesial TLE, and because the virus can induce a metabolic dysregulation that is considered to contribute to epileptogenesis, this mechanism is biologically plausible (Leibovitch and Jacobson, 2015). Interestingly, ApoE4 has been suggested to increase viral load and seizure frequency in mesial TLE patients that are positive for HHV-6B DNA and protein in temporal lobe brain samples resected during epilepsy surgery (Huang et al., 2015). Astrocytes exert many essential complex functions in the healthy CNS that are necessary to maintain synaptic and neural circuit homeostasis (Sofroniew and Vinters, 2010). Astrocytes respond to all forms of CNS insults, including viral encephalitis, through a process referred to as reactive astrogliosis, which has become a pathological hallmark of CNS structural lesions. Astrogliosis is a common step in the sequence of events that converts a normal brain into an epileptic brain after an acquired insult (Klein et al., 2018). Astrogliosis is involved in inflammatory processes as well as dysregulation of astroglial potassium and gap junction channels, which together alter glioneuronal communication and, by impairing uptake and redistribution of extracellular K+ accumulated during neuronal activity, can contribute to or cause seizures (Klein et al., 2018). Astrocytic compartmentalization of synapses also plays an essential role in neurotransmitter homeostasis by concentrating high levels of transporters for glutamate, GABA, and glycine that serve to clear these neurotransmitters from the synaptic space (Sofroniew and Vinters, 2010). During neuroinflammation, high levels of cytokines such as IL-6 lead to decreased glutamate uptake from the synaptic space by downregulating the excitatory amino acid transporter 2 (EAAT2; formerly glutamate transporter 1) on astrocytes, leading to glutamate accumulation and consequent neuronal hyperexcitability (Verhoog et al., 2020). Within this context, it is important to note that astrocytes are thought to play a crucial role in flavivirus infections of the CNS by mediating the mechanisms that underlie neurological sequelae such as seizures and epilepsy (Potokar et al., 2019; Zheng et al., 2020; Ashraf et al., 2021). Indeed, given the anatomic position of astroglia and their homeostatic role in the CNS, one can predict that virus invasion may lead to important functional consequences for the entire CNS upon the interaction of astrocytes with viruses. Furthermore, in comparison to neurons, infected astrocytes produce orders of magnitude more virus, as demonstrated for ZIKV, TBEV, and WNV (Tavkar et al., 2021). This is highly relevant for the spread of infection through the CNS, especially because astrocytes are also more resilient to the lytic effects of flavivirus infection. Interestingly, different flavivirus strains appear to exert different effects on specific astrocyte responses (Potokar et al., 2019; Ashraf et al., 2021). For example, TEBV, an important human pathogen that may result in dangerous neuroinfections (meningitis, meningoencephalitis, myelitis) and is endemic in Europe and Asia, replicates in astrocytes but does not typically affect astrocyte viability (Palus et al., 2014; Potokar et al., 2019). TBEV infection induces several morphologic and functional changes in infected rat and human astrocytes, including astrocyte activation as indicated by increased production of GFAP (Tavkar et al., 2021). Upon activation by TEBV infection, astrocytes release inflammatory cytokines and chemokines that may enhance neuronal excitability (Figure 5). TBEV infection of astrocytes may also alter the permeability of the BBB, as shown in mice (Ruzek et al., 2011). One of the key molecules that degrade the integrity of the BBB is matrix metalloproteinase 9 (MMP-9), which is overproduced in TBEV-infected astrocytes in vitro and increased in the serum and CSF of TBEV-infected patients (Potokar et al., 2019). Upon WNV infection, astrocytes also release MMPs and pro-inflammatory cytokines, leading to disruption of the BBB and recruitment of leukocytes (Ashraf et al., 2021). Analysis of autopsied neural tissues from humans with WNV encephalomyelitis revealed WNV infection of both neurons and glia (van Marle et al., 2007). In human astrocytes and neurons, WNV replicates efficiently but distinctively with a higher and faster replication rate in astrocytes (Cheeran et al., 2005). Astrocytes have an active role in the spread of WNV in the CNS and in the maintenance of WNV neuroinvasive potential. Among the WNV-induced functional changes in astrocytes is the expression of endoplasmic reticulum stress-related genes linked to WNV neurovirulence (van Marle et al., 2007). WNV-infected astrocytes also upregulate the expression of several chemokines, but only after infection with the replication-competent virus and not with an inactivated virus (Potokar et al., 2019). In an experimental murine model of WNV-induced seizures, intranasal inoculation with WNV caused limbic seizures in B6 mice, but not in IFN-γ-deficient (IFN-γ–/–) mice (Getts et al., 2007). Both strains showed similar levels of virus in the brain, as well as similar concentrations of TNF-α and IL-6, both of which alter neuronal excitability. However, TNF-α deficient mice infected intranasally with WNV still developed severe limbic seizures, similar to B6 wild-type mice (Getts et al., 2007). While the absence of seizures in the infected IFN-γ–/– mice was shown to be associated with the influence of this cytokine on excitatory circuit development, rather than a direct effect on synaptic function, per se, the observation highlights the complicated relationship between inflammation and CNS function. Finally, in patients with WNV encephalitis, increased infiltration of monocytes into the brain was found (Ashhurst et al., 2013), which, as discussed elsewhere in this review, appears to be a common outcome of CNS infection. In addition to the profound impact on fetuses and neonates (fetal growth restriction, abnormalities of the CNS, including microcephaly) caused by intrauterine infections with ZIKV during pregnancy, this virus can also cause neurologic symptoms in adults (Guillain-Barré syndrome, myelitis, encephalitis, and neuralgia) (Potokar et al., 2019). Following infection of immunocompetent pregnant mice with ZIKV, we found the virus particularly in glial cells, such as astrocytes, oligodendrocytes, and microglia, most profoundly in the brainstem and cerebellum of the maternal brain (Stanelle-Bertram et al., 2018). Interestingly, the male offspring from ZIKV infected mothers were more likely to suffer from impairment of learning and memory compared to females, likely as a result of more severe neuropathological alterations in the hippocampus compared to their female littermates (Stanelle-Bertram et al., 2018). Furthermore, in a study in which perinatal infection was simulated by using neonatal mice, seizures were observed following subcutaneous inoculation of 1-day-old immunocompetent B6 mice with ZIKV PRVABC59 (Manangeeswaran et al., 2016). The seizures were associated with ZIKV infection in the brain, neurodegeneration in the hippocampus and cerebellum, and infiltration of brain tissue with CD4+ and CD8+ T cells. In a study with ZIKV infection in 3-days-old Swiss mice, the animals developed frequent seizures during the acute phase, which were reduced by inhibiting TNF-α (Nem de Oliveira Souzaem et al., 2018). During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures and neurodegeneration, predominantly in the hippocampus, thalamus, striatum, and cortex. Both cell death and impaired proliferation of neural precursors were shown to underlie ZIKV-induced neuropathology (Nem de Oliveira Souzaem et al., 2018). In a subsequent study from the same group, the effects of ZIKV infection on neuronal networks (determined from electrophysiological activity) and how different mechanisms can trigger epilepsy in ZIKV Swiss mice were examined (Pinheiro et al., 2020). Astrocytes, together with microglia, are proposed to be major ZIKV targets in fetal brain development (Potokar et al., 2019). Primary fetal human astrocytes particularly stand out for their susceptibility to ZIKV infection in comparison with neurons and neural progenitor cells. As is the case for TBEV, astrocytes are also proposed to serve as a reservoir for ZIKV, and they apparently induce neuroinflammation through pro-inflammatory cytokines mediating synaptic and cognitive changes (Potokar et al., 2019). As with other flaviviruses, astrocytes are also an important player in altered BBB permeability in response to JEV. Upon infection with JEV, astrocytes release vascular endothelial growth factor (VEGF), IL-6, and MMPs (Potokar et al., 2019). In addition to affecting the BBB, astrocytes are also involved in neuroinflammatory responses in the JEV-infected CNS that may underlie ictogenesis. The family of small, positive-sense, single-stranded, non-enveloped RNA viruses known as the Picornaviridae includes numerous human pathogens with known and potential neurovirulence (Rotbart, 1995; Buenz and Howe, 2006), including members of the Enterovirus genus such as poliovirus, the echoviruses, the Coxsackie viruses, and the rhinoviruses. The global ubiquity of these viruses, the high transmissibility, and the widespread exposure experienced by children make picornaviruses an important component of emerging or re-emerging infections associated with neurological disease (Fischer et al., 2022). For example, enterovirus 71 (EV71), the causative pathogen in hand, foot, and mouth disease, was originally isolated in California in 1969 from a 9-month old girl with encephalitis (Schmidt et al., 1974). Further outbreaks of this and related serotypes occurred across the US, South America, Europe, and Asia, with hundreds of thousands of infections in Asia-Pacific countries since the 1990s and thousands of deaths due to encephalitis or encephalomyelitis (Puenpa et al., 2019). Notably, while seizures are reported in some of these patients (Bissel et al., 2015), a predominant outcome for children with neurologic manifestations is death, suggesting that neurovirulent picornaviruses induce severe neuropathology. As we and others have discussed, several picornavirus proteins, including the structural proteins VP1, VP2, and VP3 and the non-structural proteins 2A and 3C directly engage pro-apoptotic mechanisms in infected cells (Buenz and Howe, 2006) and co-opt antiviral mechanisms (Wang et al., 2018). However, seizures are clearly a component of picornaviral infections in less severe cases, including a broad propensity to febrile seizures, acute seizures, and late spontaneous seizures (Table 1). Picornavirus neurotropism is obviously well established for human poliovirus (Whitton et al., 2005). The human poliovirus receptor CD155 is enriched in anterior horn motor neurons (Gromeier et al., 2000) and mediates cellular entry, as proven by neuronal infection and development of paralytic poliomyelitis in mice transgenically expressing CD155 (Ren et al., 1990). Other picornaviruses exploit different cellular receptors. For example, both EV71 and coxsackievirus A16 (CVA16) utilize scavenger receptor class B, member 2 (SCARB2; aka CD36L2) to enter cells. This protein, widely and highly expressed in the brain, gut, and immune system, localizes to neurons, and transgenic expression of human SCARB2 in mice renders the host susceptible to CNS infection with EV71 (Fujii et al., 2013). While the pathophysiological relevance is not clear, it is notable that mutations in SCARB2 are associated with epilepsy (Rubboli et al., 2011). Given the broad expression of picornavirus receptors, the development of focal neurological sequelae must depend upon cell-intrinsic responses to infection or cell-specific sensitivity to innate and/or adaptive immune responses elicited by CNS infection. Concerning the former, one potential mechanism of neuronal specificity arises from the rapid and robust shutdown of host cell translation that is a hallmark of picornavirus infection (Etchison et al., 1982) and is mediated by viral protease cleavage of cap-dependent translation factor eIF-4G (Whitton et al., 2005). While cap-dependent translation is important to all cells, neurons may exhibit a unique sensitivity to translation inhibition. For example, evidence from ischemia-reperfusion models indicates that vulnerable neuronal populations in the hippocampus selectively undergo apoptosis in response to downregulated protein synthesis (Ayuso et al., 2013). Likewise, specific neuronal populations may be uniquely sensitive to the activation of stress pathways activated by translation inhibition, such as NFκB activation due to loss of IκBα translation and suppression of AKT signaling as part of the integrated stress response (Kapur et al., 2017). In parallel, suppression of glutamate transporter expression and local neuroinflammatory responses that result in the release of factors such as TNFα may combine to drive both hyperexcitability and accelerated neuronal cell death (Guerrini et al., 1995; Kaltschmidt et al., 1995; McCoy and Tansey, 2008). Finally, concerning neuron-specific sensitivity to infection-induced neuroinflammatory responses, robust evidence obtained using the mouse picornavirus TMEV, outlined below, indicates that innate immune cell-mediated acute antiviral responses lead to both neuronal cell death and dysregulation of electrophysiological homeostasis. As discussed above, animal models are useful to study the mechanisms involved in infection-induced ictogenesis (i.e., the generation of seizures) and epileptogenesis (i.e., the generation of epilepsy). Various animal species, including rabbits, rats, and mice have been infected with neurotropic viruses and develop early (encephalitis-associated) seizures, but most die following the acute viral encephalitis phase so the processes leading to epilepsy cannot be investigated (Vezzani et al., 2016). One important exception is the infection of mice with TMEV, which will be discussed in the next section. A significant advantage of animal studies is that they allow for the examination of genetic background as a variable for the host response (cf., Figure 1) to virus infection (Kollmus et al., 2018). Furthermore, animal models permit the invasive mechanistic dissection of in vivo processes underlying virus-induced CNS alterations that cannot be examined in patients. One recent example is the infection of mice with a low dose of a mouse-adapted non-neurotropic IAV (H1N1), which caused ample peripheral immune response followed by a temporary BBB disturbance (Düsedau et al., 2021). Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, a closer evaluation revealed a subtle dysbalance in glutamatergic synapse transmission in the cortex and hippocampus upon H1N1 infection. Previous experiments using IAV/H1N1 infection models have shown subtle alterations in hippocampal neuronal morphology and impairment of cognitive abilities in the absence of virus in the brain (Jurgens et al., 2012; Hosseini et al., 2018), thus demonstrating the importance of host response mediated effects as illustrated in Figure 1. In line with these findings, neuropsychiatric complications including seizures were not only reported after infection with neurotropic IAV variants but also after non-neurotropic H1N1 virus infection, especially in children (Ekstrand et al., 2010; Surana et al., 2011). A variety of animal models to study viral infections are available, including models of herpesvirus encephalitis (Reynaud and Horvat, 2013; Sehl et al., 2020), COVID-19 (Munoz-Fontela et al., 2020), ZIKV infections (Morrison and Diamond, 2017), HIV, IAV and Dengue virus infections (Krishnakumar et al., 2019), and multiple other encephalitic viruses, including JEV, WNV, and TBEV (Holbrook and Gowen, 2008). The most commonly used model species include mice, hamsters, rats, rabbits, guinea pigs, ferrets, cats, dogs, minks, pigs, chickens, ducks, fruit bats, and non-human primates. Mice have an important advantage in that the development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral pathogens and human antiviral immune responses (Lai and Chen, 2018). A recent example is the use of transgenic mice that express human ACE2 as a model for SARS-CoV-2 infection (Munoz-Fontela et al., 2020). However, with few exceptions, animal models of virus infections have not been used in the past to study the mechanisms of seizures. One explanation in this regard is that seizures, either early or late, are easily overseen if not monitored by laborious techniques, including continuous (24/7) EEG and video monitoring (Löscher, 2016). The most important example of an animal model of viral encephalitis that has been extensively used to study the molecular mechanisms of seizures and epilepsy is described in the following section. We and others have used the TMEV mouse model to study the mechanisms underlying seizure generation after virus infection of the CNS. TMEV, a non-enveloped, positive-sense, ssRNA virus of the Picornaviridae family and Cardiovirus genus, is a naturally occurring enteric pathogen of the mouse (Libbey and Fujinami, 2011). It was discovered by Nobel laureate Max Theiler in the 1930s (Theiler, 1934). TMEV causes enteric infection in mice via the fecal-oral route of transmission. While these infections are usually asymptomatic or mild, the virus can spread to the CNS and cause encephalitis and/or encephalomyelitis. Using different substrains of the virus, Theiler observed encephalomyelitis that was associated either with flaccid paralysis or seizures (Theiler, 1934; Theiler, 1937; Theiler and Gard, 1940a). He also described that the mouse virus is very rarely present in the CNS of normal mice but that intracerebral inoculation of mice with filtrates prepared from the intestinal contents of normal mice induced encephalomyelitis and the associated neurological phenotype (Theiler and Gard, 1940b). Due to the development of chronic inflammatory demyelinating disease in susceptible mouse strains such as SJL, intracerebral infection of such mice with the Daniels (DA) or BeAn 8386 (BeAn) strains of TMEV has been used as an animal model for MS for approximately the past 50 years (Libbey and Fujinami, 2021; Figure 6A). The T cell-mediated autoimmune demyelinating disease in SJL mice is characterized by weakness of the hind limbs, which advances to severe spastic paralysis, and inflammatory demyelination in the spinal cord. The B6 mouse has been used as the classic “resistant” mouse strain, which does not develop the demyelinating disease (Libbey and Fujinami, 2011). One important difference between SJL and B6 mice is that SJL mice are unable to adequately control the virus and therefore develop persistent TMEV infection that induces a smoldering neuroinflammatory environment that facilitates demyelination, particularly in the spinal cord. In contrast, the virus is rapidly cleared by B6 mice, which was thought to explain the resistance to neurological consequences of TMEV infection (Gerhauser et al., 2019; Figure 6B). The mechanisms underlying this striking difference between the strains seem to be partially due to the strong antiviral cytotoxic CD8+ T lymphocyte response that occurs in B6 mice, which is suppressed by the elevated induction of regulatory CD4+ T cells (Tregs) in SJL/J mice (DePaula-Silva et al., 2017). Theiler’s murine encephalomyelitis virus (TMEV) infection as a model for multiple sclerosis (A) or epilepsy (B). (A) Intracerebral inoculation of SJL mice with low neurovirulent Theiler’s original (TO) subgroup strains of TMEV (such as DA or BeAn) results in a biphasic disease course consisting of an acute encephalomyelitis followed by demyelination of the spinal cord. During the acute phase, neurons within the hippocampus, cerebral cortex, and spinal cord are infected. However, later, as the virus is cleared from the brain, persistent infection in spinal glia results in chronic inflammation, demyelination, axonal degeneration, and astrogliosis, resembling the pathological alterations observed in MS. Notably, seizures are not observed in TMEV-infected SJL mice at any point during the disease course. (B) Intracerebral inoculation of C57BL/6 mice with TMEV results in acute viral encephalitis that is marked by rapid monocytic and neutrophilic infiltration followed by entry of antiviral T cells, resulting in viral clearance from the host. The virus does not persist in these animals. In contrast to SJL mice, damage to hippocampal neurons is a prominent feature in B6 mice and is associated with impaired learning and memory and induction of anxiety behaviors. Early symptomatic seizures occur in ∼75% of mice during the acute phase and are associated with monocyte-derived cytokines such as IL-6 and TNF-α. The acute symptomatic seizures are observed over ∼2–10 days following infection, followed by a seizure-free latent phase of several weeks, after which about 30% of the infected mice develop spontaneous seizures. In addition to the initial description of flaccid paralysis induced by TMEV in some mice, Theiler also noted that some strains of the virus induced disease in which the “outstanding clinical sign was an extreme hyperexcitability” (Theiler and Gard, 1940a). He further noted that these animals “would jump about excitedly at the slightest stimulus,” performed “rubbing movements of the face,” and exhibited “tonic convulsions with the hind limbs extended and the fore limbs flexed.” Theiler indicated that “death might occur during one of these seizures” and that infected mice showed “marked encephalitis” in the absence of frank paralysis (Theiler and Gard, 1940a). However, this aspect of the TMEV model was largely overlooked for 50 years–regrettably, we noted seizures in one of our early studies as an exclusion criterion for behavioral assessment in B6 mice infected with the virus (Buenz et al., 2006)! It wasn’t until the foundational report from Libbey et al. in 2008 that the field came to recognize the value of the TMEV model for understanding seizures induced by viral encephalitis. These investigators reported that approximately 50% of B6 mice (male and female) infected intracerebrally with the DA strain of TMEV developed acute behavioral seizures that occurred between 3 and 10 days after virus inoculation. As with Theiler’s original observation, slight cage shaking, handling, or loud noises facilitated the occurrence of early seizures, which were rated by the Racine score (Racine, 1972). Most early seizures were generalized convulsive (Racine stage 5) seizures (Libbey et al., 2008). A similar percentage of early seizures was also observed when infecting B6 mice with the BeAn strain of TMEV (Libbey et al., 2011). The incidence of such seizures increased to 75% if continuous video-EEG was used to monitor the mice (Stewart et al., 2010a). Furthermore, Stewart et al. (2010a) reported that a significant proportion of mice experiencing acute seizures later developed spontaneous epileptic seizures with hippocampal sclerosis (Figure 6B), which is a hallmark of TLE (Blümcke et al., 2002; Thom, 2014). In B6 mice, TMEV has a specific tropism for the CA1 and CA2 pyramidal cell layers of the hippocampus; periventricular thalamic nuclei; septal nuclei; and piriform, parietal, and entorhinal cortices during acute TMEV infection (Libbey et al., 2008; Buenz et al., 2009; Stewart et al., 2010a,b). Unlike B6 mice, TMEV-infected SJL mice show subclinical, transient polioencephalitis along with mild neuronal degeneration, which is not accompanied by seizure development in the acute disease (Libbey et al., 2008; Figure 6A). SJL mice are typically protected from hippocampal damage by TMEV, which seems to be mediated by IL-10 receptor signaling (Uhde et al., 2018). Theiler’s murine encephalomyelitis virus infection in B6 mice was the first animal model to associate viral encephalitis with epilepsy, thus allowing the field to study the mechanisms underlying the development of early and late seizures. Based on a large series of subsequent studies of the groups of Robert S. Fujinami, H. Steve White, and Karen S. Wilcox at the University of Utah, which were reviewed by Libbey and Fujinami (2011) and DePaula-Silva et al. (2017, 2021), it was suggested that infiltrating monocytes (CD45hi CD11b+) present in the brain of B6 mice at day 3 post-infection are an important source of IL-6, which critically contributes to the development of acute seizures in the TMEV-induced seizure model. Furthermore, the production of high levels of TNF-α by microglia during the acute phase of the infection was found to play a role (Cusick et al., 2013). When mice deficient in TNF receptors, TNF-α or IL-6 were infected with TMEV, the incidence of acute seizures was significantly decreased, whereas IL-1R1 deficient mice did not differ from wild-type controls (Kirkman et al., 2010; Patel et al., 2017). From these data and the known effects of IL-6 and TNF-α on neuronal activity, it was suggested that IL-6 and TNF-α secreted in the brain by infiltrated monocytes and resident microglia during TMEV infection in B6 mice may contribute to enhanced glutamatergic excitation and decreased GABAergic inhibition and lead to a more seizure prone state (DePaula-Silva et al., 2021). In support of this hypothesis, TMEV infection of B6 mice depleted of monocytes resulted in a significant decrease in the number of mice experiencing seizures, substantiating a role for infiltrating monocytes in the development of acute seizures in the TMEV-induced seizure model (Cusick et al., 2013). However, at least in part, the experimental methods used to reduce monocyte invasion and distinguish monocytes/macrophages from microglia were not specific, so the exact role and interplay of these and other immune cells in the TMEV model remained elusive. The interesting data reported by the University of Utah groups prompted W. Löscher’s group to establish the TMEV model in B6 mice in Hannover, Germany. Unexpectedly, it took several years to reproduce the seizure phenotype in our laboratory (Bröer et al., 2016). Indeed, the BeAn strain of TMEV was used in thousands of SJL/J and B6 mice by Wolfgang Baumgärtner’s group at the Department of Pathology at the University of Veterinary Medicine in Hannover over a period of ∼15 years in numerous studies on mechanisms involved in virus-induced demyelination but seizures were never observed in B6 mice. Thus, we hypothesized that either the substrain of B6 mice or the BeAn substrain used in these experiments may have been responsible for the lack of seizures. This hypothesis was addressed by comparing two B6 and two BeAn substrains, including the mouse and virus substrains used in the original studies of Fujinami and White (Bröer et al., 2016). In addition, we compared the potency of the BeAn and DA TMEV strains to induce seizures and epilepsy in mice. The idea behind this approach was to study what is and what is not necessary for the development of acute and late seizures after brain infection in mice. Receiver operating characteristic (ROC) curve analysis was used to determine which virus-induced brain alterations are associated with seizure development. In B6 mice infected with different TMEV virus (sub)strains, the severity of hippocampal neurodegeneration, amount of MAC3-positive microglia/macrophages, and expression of ISG15 were almost perfect at discriminating seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor (Bröer et al., 2016). The potential role of blood-borne monocyte brain invasion for the seizure phenotype induced by TMEV infection of B6 mice suggested by the University of Utah groups (Libbey and Fujinami, 2011; DePaula-Silva et al., 2017, 2018, 2021) prompted us to perform a series of studies using either genetic or pharmacological strategies. The outcome of these studies is summarized in Table 3. First, to better differentiate brain-resident myeloid cells, including microglia, from invading monocytes in the TMEV encephalitis model of TLE, we compared virus-induced effects in B6 WT vs. B6-based Cx3cr1-creER±tdTomatoSt/Wt mice, in which long-lived CX3CR1+ cells such as microglia can be distinguished from infiltrating monocytes by the expression of the red fluorescent protein tdTomato (Käufer et al., 2018a). When using flow cytometry to differentiate blood-borne monocytes (CD45highCD11b+) from resident microglia (CD45lowCD11+) in the brain, the Cx3cr1-creER±tdTomatoSt/Wt reporter mice provided qualitative proof that activated myeloid cells present in the CNS after TMEV infection consist of microglia and infiltrating monocytes (Table 3), although concerning CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection (Käufer et al., 2018a). A summary of the experiments of the Löscher lab on TMEV-induced seizures and epilepsy. Data are from TMEV-infected C57BL/6 mice, using the DA strain of TMEV for intracerebral inoculation. In addition to data from flow cytometry shown in the table for monocyte invasion and microglia proliferation, neuroinflammation was also assessed by immunohistochemistry (Iba1, Mac-3, CD3), T cell and neutrophil infiltration, and qPCR (cytokines). A significant increase in infected wild-type controls or Cx3cr1 reporter mice compared to sham-infected controls is indicated by “+” and lack of such alteration by “Ø”. A significant decrease or increase by pharmacological or genetic manipulation compared to infected wild-type controls is indicated by arrows. TBD, to be determined. Next, we used two pharmacological approaches to determine the impact of invading monocytes vs. resident microglia for early seizures and hippocampal damage induced by TMEV in B6 mice. When using systemic administration of liposome-encapsulated clodronate liposomes as a selective and widely used approach for monocyte depletion, almost complete depletion of monocytic cells was achieved in the spleen and blood of Theiler’s virus-infected B6 mice, which was associated with a 70% decrease in the number of brain-infiltrating monocytes as assessed by flow cytometry (Waltl et al., 2018a). As shown in Table 3, significantly fewer clodronate liposome-treated mice exhibited seizures than liposome controls. The severity of seizures was not affected by monocyte depletion, but the seizure burden (the number of seizures per mouse observed over 7 days after infection) was markedly reduced (Waltl et al., 2018a). However, the development of hippocampal damage was not prevented or reduced by monocyte depletion (Table 3). Surprisingly, clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage (Waltl et al., 2018a). Thus, our next pharmacological approach used prolonged administration of PLX5622, a specific inhibitor of colony-stimulating factor 1 receptor that depletes microglia (Waltl et al., 2018b). As shown in Table 3, microglia depletion accelerated the occurrence of seizures, exacerbated hippocampal damage, and led to neurodegeneration in the spinal cord, which is normally not observed in B6 mice. These data suggested that microglia are required early after infection to limit virus distribution and persistence, most likely by modulating T cell activation (Waltl et al., 2018b). An antiviral role of microglia has also been demonstrated for ZIKV, HSV, JEV, WNV, and several other virus infections (Terry et al., 2012; Chen et al., 2019). Interestingly, TNF-α expression in the brain of TMEV-infected mice was not affected by microglia depletion, suggesting that CNS and/or infiltrating cells other than microglia are also secreting this cytokine (Waltl et al., 2018b). More recently, our data have been partially confirmed by the University of Utah groups (DePaula-Silva et al., 2018; Sanchez et al., 2019). In an additional series of experiments, we used genetic approaches (Ccr2-KO and Cx3cr1-KO mice) to study the role of invading monocytes vs. activated microglia for early seizures and hippocampal damage (Käufer et al., 2018a). CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation (Prinz and Priller, 2010). Based on their differential expression of the chemokine receptors CCR2 and CX3CR1 in mice, so-called “inflammatory” (or “classic”) monocytes (CCR2+CX3CR1low), which are highly mobile and rapidly recruited to inflamed tissues, can be distinguished from patrolling (non-classic) monocytes (CCR2–CX3CR1high), which are larger in size and patrol along vascular endothelium such as the BBB (Prinz et al., 2011; Prinz and Priller, 2017). Brain-resident microglia produce the myelo-attractant cytokine CCL2 (also known as MCP1), a CCR2 ligand that promotes the transmigration of CCR2+ monocytes (and T cells) across the BBB via CCL2/CCR2 crosstalk (Prinz and Priller, 2010; Howe et al., 2017). Mice devoid of the Ccr2 gene exhibit markedly reduced recruitment of monocytes and reduced pathology in several brain disease models, including autoimmune encephalitis, MS, stroke, and status epilepticus (Prinz and Priller, 2010; Chu et al., 2014; Varvel et al., 2016). Interestingly, in SJL mice, in which infection with TMEV induces severe spinal cord demyelination (Figure 6A), the use of Ccr2-KO mice reduced monocyte infiltration, demyelination, and long-term disease severity (Bennett et al., 2007). As shown in Table 3, in B6 mice, the lack of CCR2 or CX3CR1 receptors was associated with a significant reduction of monocyte invasion and almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection (Käufer et al., 2018a). These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis. It is also important to note that the consequences of pharmacological vs. genetic manipulation of monocyte invasion and microglia activation strikingly differed (Table 3). Furthermore, the interplay between microglia and invading monocytes in this model is more complex than previously proposed by other groups (Libbey and Fujinami, 2011; DePaula-Silva et al., 2017, 2018, 2021). All studies described thus far examined the role of various manipulations on the occurrence of early seizures and hippocampal damage in the TMEV model in B6 mice. As described above, a fraction of the mice also develops spontaneous recurrent seizures, i.e., epilepsy after a latent period of several weeks (Figure 6B). In the experiments of the Löscher group, the incidence of epilepsy was determined by continuous (24/7) video-EEG monitoring, resulting in an epilepsy incidence of 33%, while the incidence of early seizures was 77% (Anjum et al., 2018). When determining the development of epilepsy in mice following treatment with clodronate liposomes or in Ccr2-KO and Cx3cr1-KO mice, no significant difference from controls was observed (Käufer et al., 2018b; Waltl et al., 2018c). This would suggest that–as outlined above–the mechanisms underlying early and late seizures are different. In this respect, it is interesting to note that although there are significant increases in amplitude and frequency of spontaneous and miniature excitatory currents (mediated by glutamate) in hippocampal CA3 neurons recorded in brain slices prepared during the acute infection period and during chronic epilepsy 2 months after infection, the patterns of changes observed are markedly different during these two periods, suggesting that there are underlying changes in the network over time (Smeal et al., 2012). In addition to the changes in excitatory currents of CA3 neurons both during the acute infection and 2 months later shown by Smeal et al. (2012), additional experiments disclosed a decrease in CA3 inhibitory network activity (mediated by GABA) during the acute infection, but not at the 2-month time point, again suggesting different mechanisms of seizure generation during the acute infection and during chronic epilepsy (Smeal et al., 2015). In addition to epilepsy as a long-term outcome of TMEV infection in B6 mice, these animals also exert behavioral and cognitive alterations, such as increased anxiety, decreased pentylenetetrazole seizure threshold, and impaired learning and memory (Umpierre et al., 2014; Barker-Haliski et al., 2015). Treatment of mice with minocycline, but not valproic acid, during the acute phase of the TMEV infection improved long-term behavioral outcomes in the TMEV model (Barker-Haliski et al., 2016), but epilepsy was not monitored in this study. Minocycline was used in this study to directly suppress microglial activation and overexpression of inflammatory cytokines. In summary, our data on the TMEV model suggest that hippocampal damage is not critically involved in ictogenesis and epileptogenesis, because genetic manipulations that completely prevented the damage did not modify the incidence of early or late seizures (Käufer et al., 2018a,b). The mismatch between findings of genetic versus pharmacological manipulations in TMEV-infected B6 mice illustrated in Table 3 deserves further study. In parallel with the elegant work of the Utah and Hannover groups, the Howe group also came to recognize the importance of infiltrating inflammatory monocytes. Our initial studies probed the neuropathological and behavioral sequelae of TMEV encephalitis in B6 mice, revealing that pyramidal neurons in the CA1 region of the hippocampus were selectively lost by 4 days after intracerebral inoculation with Daniel’s strain of TMEV and that mice tested in the Morris water maze starting at 11 days after infection exhibited a profound disruption in the ability to form spatial memories (Buenz et al., 2006). We showed that memory impairment was associated with damage to the CA1 region in two ways. First, the increasing hippocampal injury was associated with a graded loss in the ability to learn the maze; second, mice with any amount of hippocampal damage converted from a spatial memory strategy to a cue-based escape strategy. At the time, we focused on the role of neurotropic viruses in the direct killing of hippocampal neurons (Buenz and Howe, 2006) and we postulated that low-level neurovirulence amongst the human picornaviruses results in widespread erosion of cognitive reserves in humans, potentially explaining the development of memory and cognitive impairments with age in the absence of clear etiology. Unexpectedly, however, our follow-up studies indicated that apoptosis of hippocampal neurons during acute TMEV encephalitis occurred independently of direct cellular infection (Buenz et al., 2009). Indeed, while many infected mice exhibited nearly complete loss of all CA1 neurons in the dorsal hippocampus, only a small fraction of these neurons expressed TMEV antigen before death. This is consistent with our contention that only 20–2000 cells in the brain are infected with TMEV in the immediate aftermath of inoculation (Howe et al., 2017). Moreover, we showed that CA1 neurons exhibited evidence of oxidative injury and apoptotic processes as early as 2 days after inoculation, with peak neuronal death occurring within 4 days, a timeline that is inconsistent with any effect of antiviral adaptive immune-mediated mechanisms (Dethlefs et al., 1997; Mendez-Fernandez et al., 2003). Given the virus-independent nature of the CA1 pyramidal neuron death, we next sought to protect these neurons by interfering with the apoptotic cascade. We observed that calpain was specifically activated in CA1 neurons as early as 2 days after TMEV inoculation, prompting us to treat infected animals with ritonavir, a drug designed as an HIV protease inhibitor that also suppresses calpain (Howe et al., 2016). We found that ritonavir therapy almost completely prevented the loss of CA1 pyramidal neurons, without impacting viral fitness or eventual viral clearance. Moreover, we found that calpain inhibition preserved cognitive performance in the Morris water maze, protected novel object recognition learning, and completely prevented the development of acute, high Racine score seizures. Critically, this therapeutic effect was achieved even when therapy was started at 36 h after inoculation, a timepoint at which mice already exhibit low Racine score events and encephalitis is well established (Howe et al., 2016). In parallel with the ritonavir work, we published two studies showing that inflammatory monocytes are the primary driver of hippocampal injury and cognitive impairment in the TMEV model. In the first, we showed that inflammatory monocytes infiltrate the TMEV inoculated brain within hours (Howe et al., 2012a)–indeed, in our most recent work we have observed these cells in the hippocampus within 3 h of inoculation. We defined these cells as CD45hiCD11b+ cells that are positive for Ly6C and Ly6B but negative for Ly6G. We also established that the LysM:GFP mouse generated by David Sacks (Faust et al., 2000) (not based on the LysM-Cre line) permitted the clear delineation of infiltrating inflammatory monocytes (GFPmid), infiltrating neutrophils (GFPhi), and microglia (GFPneg). Furthermore, we showed that immunodepletion of monocytes but not neutrophils preserved cognitive performance in the Morris water maze and protected the hippocampus from injury. In the second study, we showed that despite equivalent viral load and acute encephalitis, SJL mice do not exhibit any injury to the hippocampus and this effect was genetically dominant, as the F1 offspring of SJL × B6 mice also showed hippocampal preservation (Howe et al., 2012b). Relevant to the discussion above regarding the direct viral killing of neurons, we also observed large numbers of intact CA1 pyramidal neurons loaded with TMEV antigen at 3 days after inoculation; these neurons eventually clear the virus non-lytically and remain intact. Strikingly, we found that SJL mice exhibited a markedly truncated inflammatory monocyte response at 24 h after inoculation, while neutrophil infiltration levels were the same or greater than B6 mice at the same timepoint. The B6 response profile was recapitulated in B10.S mice (Patick et al., 1990) (a C57BL/10 congenic line that expresses H-2s and is therefore histocompatible with SJL mice) and we used bone marrow reconstitution to create chimeric animals with a B10.S nervous system and SJL immune system or an SJL nervous system with a B10.S immune system. We found that reconstitution of SJL mice with a B10.S immune system resulted in robust inflammatory monocyte infiltration and consequent hippocampal injury that was indistinguishable from B10.S mice reconstituted with B10.S bone marrow. Finally, we showed that adoptive transfer of Ly6C+Ly6G– B6 peritoneal exudate monocytes (induced by mineral oil) into B6 × SJL F1 hosts at 18 h after TMEV inoculation led to profound hippocampal injury and abrogation of scent-based novel object recognition learning. Given the acute timing of the inflammatory monocyte response and the rapid initiation of hippocampal injury and behavioral seizures in B6 mice, we sought to identify the molecular and cellular sources driving leukocyte recruitment to the CNS. We found that by 3 h after intracranial inoculation of TMEV the hippocampus exhibited a profound upregulation of inflammatory chemokine transcripts that was quickly followed by upregulation of inflammatory cytokine RNA (Howe et al., 2017). Moreover, we observed that serum CCL2 levels peak at 3 h after infection and this was temporally associated with high levels of CCL2 in the brain and hippocampus. Genetic deletion of CCR2 essentially abrogated inflammatory monocyte infiltration, while systemic immunodepletion of CCL2 but not CCL7 also truncated the monocytic response during acute TMEV encephalitis. Unexpectedly, we found that CCL2 was predominantly expressed by hippocampal neurons at 6 h after TMEV inoculation and we showed that neuron-specific deletion of CCL2 (Syn-Cre × CCL2-RFPfl/fl) resulted in complete suppression of serum and hippocampal CCL2 levels at this timepoint and greatly attenuated inflammatory monocyte infiltration at 24 h after inoculation. Finally, we have recently determined that the size of the inflammatory monocyte response during acute TMEV encephalitis effectively controls the extent of hippocampal injury, the loss of spatial learning, and the induction of high-grade Racine score behavioral seizures in B6 mice (Howe et al., 2022). In this work, we used different amounts of initial TMEV inoculum to drive different levels of encephalitis. We found that introducing 12,500 plaque-forming units of TMEV into the brain elicited encephalitis at 24 h, which was 90% less intense than our standard inoculum of 200,000 plaque-forming units in terms of absolute numbers of infiltrating inflammatory monocytes. While at first glance this seems obvious, it is critical to note that at 24 h the total load of infectious virus in the brain was equivalent between the two inocula. This means that the initial viral exposure, not the amount of replicated virus, set the pace for the downstream encephalitic response. Indeed, animals inoculated with the lower amount of virus exhibited essentially no increase in CCL2 at 24 h and exhibited no increase in TNFα or IL6 in the hippocampus at 24 or 72 h after inoculation. These mice had limited hippocampal injury and showed complete preservation of spatial learning in the Barnes maze. Assessment of behavioral seizures through the first 10 days after inoculation revealed that the low virus group exhibited no high-level Racine seizures at any timepoint. EEG analysis confirmed reduced ictal activity. Notably, however, the low virus group still developed low-grade Racine seizures and did exhibit EEG abnormalities. Looking at microglial activation in these animals, we found that there was equivalent upregulation of Iba-1+ microglia in the hippocampus between viral inocula and these microglia showed equivalent upregulation of activation markers such as CD44. Within this context, we also found that mice inoculated with the lower amount of TMEV were more resistant to kainic acid-induced status epilepticus at 24 h relative to mice receiving the standard inoculum, but were still more sensitive than uninfected mice. These findings suggest that microglial activation acts as a binary switch during acute CNS viral infection while the infiltrating monocyte response (and encephalitis, stricto sensu) is graded. Moreover, while microglial activation during CNS viral infection primes the brain for ictogenesis, the full induction of acute clinical-grade seizures requires infiltration of inflammatory monocytes. This may have profound implications for considerations of ictogenesis during viral encephalitis. For example, even a small amount of viral invasion into the CNS may trigger microglial activation that confers a decrease in seizure threshold without rising to the level of clinical manifestations. In the context of a patient with other factors predisposing to ictogenesis, this microglial effect may be sufficient to push the system over into a seizure state. Likewise, the critical role of inflammatory monocyte infiltration in driving seizures during viral encephalitis provides an opportunity to consider therapeutic approaches that prevent these cells from invading the CNS. In numerous experiments over many years, we have never observed a detrimental effect on viral clearance associated with suppression of inflammatory monocyte responses, while we have repeatedly observed neuroprotective effects of reducing inflammatory monocyte infiltration. This leads us to strongly favor the development of new therapies to inhibit these cells or the application of unconventional therapies such as monocyte adsorption apheresis in patients with viral encephalitis. Following the acute innate response to intracerebral inoculation with TMEV, a robust adaptive response is mounted, leading to infiltration of virus-specific CD8+ cytotoxic T lymphocytes, which play a significant role in viral clearance from the host (Libbey and Fujinami, 2011). In B6 mice, the earliest wave of anti-viral T cells arrives in the brain around 4 days after inoculation, peaking around day 7 (Deb and Howe, 2008). This response is marked by nearly complete restriction to recognition of a peptide derived from the VP2 capsid protein presented on the Db MHC class I molecule (Johnson et al., 1999; Howe et al., 2007). While antiviral CD8+ T cells recognize and kill infected cells, within the CNS it is vital to host survival and function to clear virus non-lytically via mechanisms such as IFNγ (Rodriguez et al., 2003). The involvement of CD8+ cytotoxic T lymphocytes and viral clearance in the development of acute seizures in the TMEV-induced seizure model was assessed through the use of OT-I transgenic mice (B6 background), in which the majority of the CD8+ T cells carry an ovalbumin-specific T-cell receptor (Kirkman et al., 2010). The number of TMEV-infected OT-I mice experiencing acute seizures was comparable to wild-type B6 mice, suggesting that the seizures were not influenced by TMEV-specific CD8+ T cells. In TMEV-infected B6 mice, the acute symptomatic seizures resolve by ∼day 10 post-infection (Figure 6B), which was also observed in OT-I mice, indicating that the cessation of seizures was also not due to the clearance of virus by the CD8+ T-cell response (Kirkman et al., 2010). Similarly, in our studies, we found no significant correlation (by ROC analysis) between T lymphocyte brain infiltration and acute symptomatic seizures (Bröer et al., 2016). More recently, RAG1–/– mice, which are deficient in mature T and B cells, were compared with B6 mice infected with TMEV (DePaula-Silva et al., 2018). As expected, CD4+ and CD8+ T cells were absent from the brains of RAG1–/– mice, but the number of RAG1–/– mice experiencing seizures was similar to control mice, further substantiating that lymphocytes are not playing a role in the development of acute seizures following TMEV infection (DePaula-Silva et al., 2018). When we depleted microglia by prolonged treatment with PLX5662 from 21 days before to 6 or 7 days after TMEV infection of B6 mice, an unfavorable hippocampal and spinal cord ratio between Tregs and effector T cells was observed, thus reducing antiviral immunity in these regions (Waltl et al., 2018b). This possibility was substantiated by a marked increase in brain mRNA expression of the immunosuppressive cytokine IL-10 in the brain of infected PLX5622-treated mice, which is released by Tregs and suppresses the activation of cytotoxic T cells. These data thus added to the concept of microglia–T cell crosstalk (Schetters et al., 2017). Recently, it has been proposed that a dysregulated microglia-T-cell interplay during viral infection may result in altered phagocytosis of neuronal synapses by microglia that causes neurocognitive impairment (Chhatbar and Prinz, 2021). The role of glutamate receptors and transporters in the TMEV model in B6 mice was studied by the University of Utah groups. Based on previous data from these groups indicating that the inflammatory cytokines IL-6 and TNF-α play a role in seizure development in the TMEV model and that infiltrating monocytes are major producers of these cytokines, the potential role of the metabotropic glutamate receptor 5 (mGluR5) was examined (Hanak et al., 2019). mGluR5 is a G-protein coupled receptor that has been shown to reduce IL-6 and TNF-α production in microglia and macrophages and to provide neuroprotection in other disease models (Loane et al., 2014; Zhang et al., 2015). Hanak et al. (2019) found that pharmacological stimulation of mGluR5 with the selective positive allosteric modulator VU0360172 not only reduced acute seizure outcomes in TMEV-infected B6 mice but also reduced the percent of microglia and macrophages producing TNF-α 3 days post-infection. Immunofluorescence confocal imaging showed a significant decrease in mGluR5 immunoreactivity in the CA1 and CA3 regions of the hippocampus with no significant changes seen in the dentate or cerebral cortex (control brain region) in TMEV-infected B6 mice with seizures compared to controls (Hanak et al., 2019). Concerning ionotropic glutamate receptors, i.e., NMDA, kainate, and AMPA receptors, Libbey et al. (2016) determined the effects of three antagonists, MK-801, GYKI-52466, and NBQX, on acute seizure development in the TMEV-induced seizure model in B6 mice. Surprisingly, they found that only the AMPA receptor antagonist NBQX affected acute seizure development, resulting in a significantly higher number of mice experiencing seizures, an increase in the number of seizures per mouse, a greater cumulative seizure score per mouse, and a significantly higher mortality rate among the mice. This proconvulsant effect of NBQX observed in the TMEV-induced seizure model was unexpected, because NBQX has previously been shown to be a potent anticonvulsant in a variety of animal seizure models (Catarzi et al., 2007). In another study, the role of glutamate transporters was examined (Loewen et al., 2019). Glutamate transporters such as GLT-1 expressed by glial cells contribute significantly to the control of extracellular glutamate levels, and the expression profile and function of these glutamate transporters have been implicated in epilepsy (Peterson and Binder, 2020). TMEV-infected seizing B6 mice show evidence of reactive astrogliosis, which has been associated with decreases in glutamate transporter expression and function in sclerotic tissue (Proper et al., 2002; Dossi et al., 2018). However, pharmacological and genetic methods used to modulate the glial glutamate transporters, while effective in other models, were not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected B6 mice (Loewen et al., 2019). Overall, the TMEV encephalitis model of acute and late seizures in B6 mice is a powerful tool for testing the inflammatory mechanisms that drive ictogenesis. In addition to providing a robust platform for manipulating the host immune response during viral encephalitis to alter seizure biology, the TMEV model also serves as a new, biologically and clinically relevant platform for testing established and novel therapeutics in the context of seizures that evolve without the introduction of ictogenic pharmacological agents or electrical stimulation (Metcalf et al., 2021). While a tremendous amount of progress has been made in understanding the acute phase of TMEV encephalitis, much work remains to discover the cellular and molecular mechanisms that link inflammation to ictogenesis and, critically, to identify the mechanisms that lead from the acute phase of the disease to the development of late, spontaneous seizures (DePaula-Silva et al., 2021). Both the early (acute symptomatic) seizures and the late (spontaneous recurrent) seizures occurring in the acute and chronic phases of the infection, respectively (Figure 4), can be symptomatically suppressed in many patients using antiseizure medications (ASMs) such as levetiracetam, phenytoin, and others (Löscher and Klein, 2021a). More than 30 ASMs are clinically approved; adequate choice of treatment depends on a variety of factors, including the type of seizures and epilepsy (Löscher and Klein, 2021a). However, about 30% of patients with epilepsy do not respond adequately to ASM treatment (Janmohamed et al., 2020), indicating that more research is required to identify the multiple mechanisms driving seizures associated with viral encephalitis and other brain insults. In principle, the use of ASMs does not differ between early and late seizures, but control of acute symptomatic seizures during viral infection requires simultaneous treatment of the underlying etiology (Koppel, 2009; Gunawardane and Fields, 2018; Löscher and Klein, 2021a). Preferred medications for the treatment of acute symptomatic seizures or status epilepticus are those available for intravenous use, such as benzodiazepines, fosphenytoin or phenytoin, valproate, levetiracetam, and phenobarbital (Gunawardane and Fields, 2018). Prophylactic treatment with ASMs should be started as soon as possible after the onset of infection and continued as long as needed (Singhi, 2011). If not adequately treated, early seizures may progress to status epilepticus. However, prophylactic ASMs are not administered in all cases after onset of infection. Instead, ASMs are often withheld until the first-post-infection seizure. Although the use of ASMs after the onset of infection appears clinically plausible, there is insufficient evidence to support or refute the routine use of ASMs for the prevention of early seizures in viral encephalitis. Concerning other causes of early seizures, only a few placebo-controlled clinical studies on the treatment of early seizures exist. In a study in which treatment with intravenous phenytoin or placebo was started within 24 h of traumatic brain injury, 3.6% of the patients assigned to phenytoin had seizures between the onset of treatment and day 7, as compared with 14.2% of patients assigned to placebo (P < 0.0001; Temkin et al., 1990). Although phenytoin is the standard of care to prevent acute symptomatic seizures after brain injury, a recent meta-analysis of clinical studies indicated that levetiracetam has similar efficacy to phenytoin in preventing such seizures (Zhao et al., 2018). Concerning the treatment of febrile seizures in children, most febrile seizures are self-limited (simple febrile seizures); however, when seizures last longer than 5 min (complex febrile seizures or fSE), a benzodiazepine should be administered to break the seizure (Löscher and Klein, 2021a). A 2018 Cochrane review concluded that intravenous lorazepam and diazepam have similar rates of seizure cessation and respiratory depression (McTague et al., 2018). When intravenous access is unavailable, buccal midazolam or rectal diazepam is acceptable. In contrast to febrile seizures, FIRES is very difficult to treat. Treatment modalities for these patients include, among others, various ASMs, ketogenic diet, intravenous corticosteroids, intravenous immunoglobulin, and burst-suppression coma (Hon et al., 2018). More recently, based on our initial report of efficacy in a child with FIRES (Kenney-Jung et al., 2016; Clarkson et al., 2019), the IL-1R antagonist anakinra has been increasingly used in the treatment of these patients, with mixed results (Farias-Moeller et al., 2018; Löscher and Klein, 2021a; Yamanaka et al., 2021). Furthermore, the IL-6 receptor antagonist tocilizumab has been used as an alternative (Stredny et al., 2020; Yamanaka et al., 2021). In the TMEV model in B6 mice, the efficacy of various ASMs and anti-inflammatory compounds to suppress acute symptomatic seizures has been tested. In studies by Barker-Haliski et al. (2015, 2016), valproate, but not carbamazepine or minocycline, reduced the acute seizure burden. In a recent study by Metcalf et al. (2021), several prototype ASMs were effective, including lacosamide, phenytoin, ezogabine, phenobarbital, tiagabine, gabapentin, levetiracetam, topiramate, and valproate. Of these, phenobarbital and valproate had the greatest effect (>95% seizure burden reduction). The prototype anti-inflammatory drugs celecoxib, dexamethasone, and prednisone also moderately reduced seizure burden. Furthermore, cannabidiol reduced seizures in the TMEV model (Patel et al., 2019). The TMEV model in B6 mice is currently utilized by the NIH/NINDS-funded Epilepsy Therapy Screening Program (ETSP) as a tool for evaluating the antiseizure effect of novel compounds (Wilcox et al., 2020). However, it is important to note that transient treatment of acute symptomatic seizures with ASMs has no proven effect on the subsequent development of epilepsy (Löscher and Klein, 2021a). Similarly, it is not entirely clear whether treatment of FIRES with anakinra improves the long-term prognosis of affected patients (Yamanaka et al., 2021), though several studies report adequate seizure control with ASMs as long as anakinra therapy is maintained (Kenney-Jung et al., 2016; Yamanaka et al., 2021). Prevention of epilepsy after neurological insults such as viral encephalitis is an unmet clinical need (Pitkänen and Lukasiuk, 2011; Löscher et al., 2013; Löscher and Klein, 2021a). In principle, there are at least three strategies to prevent epilepsy after brain infections or other epileptogenic brain insults (Vezzani et al., 2016), (1) prevention of the initial insult; (2) initial insult modification (diminishing the long-term consequences of the insult by reducing the severity or duration of the initial brain insult); and (3) “true” antiepileptogenesis or disease modification after the insult by interfering with the mechanisms underlying epileptogenesis. Ad 1, wearing masks in public, maintaining a safe distance from others, and getting vaccinated are examples of prevention of the initial insult, i.e., the infection. Ad 2, intervention with appropriate treatment of the CNS infection that modifies the initial insult and thereby reduces the risk of long-term consequences. A novel emerging strategy is targeting phosphatidylserine receptors such as the TAM tyrosine kinase receptor family (TYRO3, AXL, and MERTK), which confers potent protection against neuroinvasion and CNS lesion development during neuroinvasive virus infection (Wang et al., 2021b). Our findings showing preservation of the hippocampus and inhibition of ictogenesis in mice treated with a calpain inhibitor during the acute phase of TMEV encephalitis is another potential example (Howe et al., 2016). Ad 3, antiepileptogenesis or disease modification after the infection includes treatments that directly target the complex mechanisms underlying epileptogenesis and/or actively repair damaged neural circuits. As discussed above, the available experimental evidence supports the idea that inflammation in the brain caused by viral infections may contribute to acute seizures and epilepsy development using partially overlapping mechanisms. This highlights the possibility of identifying common targets for therapeutic interventions which may not only suppress the symptoms of the disease but also interfere with key pathogenic mechanisms. Thus, one can envisage the use of specific anti-inflammatory drugs blocking the key pathogenic inflammatory mechanisms (Vezzani, 2014, 2015). The advantage of this approach is that some of these drugs are already available in the clinic for the treatment of auto-inflammatory or autoimmune diseases, such as the IL-1R antagonist anakinra, the IL-1α antagonist canakinumab, the IL-6 receptor antagonist tocilizumab, and the TNF-α antibody infliximab or the TNFα receptor fusion protein etanercept. Preclinical research is important to determine the therapeutic potential of such novel treatments. The challenge, however, is to design an intervention that blocks the detrimental arm of brain inflammation without interfering with the homeostatic mechanisms; in this context, the implementation of resolving anti-inflammatory mechanisms rather than the prevention of the inflammatory cascade may be a better strategy to avoid complications surrounding viral control and clearance (Vezzani et al., 2016). Preventing inflammation may also be difficult due to the rapid onset and amplification of the pathogenic cascade after the first inciting event. Animal models are important in the search for novel treatments that provide disease-modifying efficacy after viral infections. In a study by Barker-Haliski et al. (2016), in which TMEV-infected B6 mice were treated during the acute phase with minocycline to suppress microglial activation and overexpression of inflammatory cytokines, minocycline improved long-term behavioral outcomes and normalized seizure threshold. However, because late spontaneous seizures are relatively rare in the TMEV model (Figure 6B), necessitating continuous (24/7) video-EEG monitoring of large groups of infected mice, the potential effect of minocycline on the development of epilepsy was not examined by Barker-Haliski et al. (2016). As discussed above, we used continuous video-EEG monitoring in the TMEV model in B6 mice to determine the potential antiepileptogenic effect of pharmacologic and genetic manipulations interfering with monocyte invasion and found no significant effects on the development of epilepsy (Käufer et al., 2018b; Waltl et al., 2018c). The disparity between this finding and evidence that inflammatory monocytes contribute to early ictogenesis further underscores the need for more extensive research in this arena. Febrile seizures in children often, but not always, occur in the context of an ongoing systemic virus or bacterial infection (Vezzani et al., 2016). This clinical setting has been reproduced in immature rodents by systemic administration of lipopolysaccharide (LPS) to mimic Gram-negative infections or poly(I:C) to mimic viral infections. This acute challenge imposed in a specific developmental window (postnatal day 7–14) increases the susceptibility to subsequent seizures induced by kainate, pilocarpine, or pentylenetetrazole (Galic et al., 2009; Riazi et al., 2010; Galic et al., 2012). The increased susceptibility to provoked seizures was maintained in the animals until adulthood. Systemic inflammation, not the fever per se, transiently induced IL-1α and TNF-α in the hippocampus and neocortex, and prevention of this brain response with minocycline, or using an IL-1R antagonist (anakinra) or TNF-α inactivating antibodies, precluded both the acute and long-term reduction in seizure threshold, as well as the comorbidities (anxiety-like behavior and learning and memory deficits) observed in adulthood (Riazi et al., 2010; Galic et al., 2012). The transient inflammatory challenge permanently altered the expression of glutamate receptor subtypes and the Na-K-2Cl co-transporter NKCC1 in the rat forebrain, which may have implications for the observed long-term pathophysiological outcomes (Reid et al., 2013; Riazi et al., 2015). Interestingly, transient treatment with anakinra after brain injury has been reported to provide disease-modifying or antiepileptogenic effects in non-infectious post-status epilepticus models of acquired epilepsy (Noé et al., 2013; Dyomina et al., 2020). Similarly, the immunomodulator fingolimod, which is established as an MS therapy, was reported to exert antiepileptogenic, neuroprotective effects, and anti-inflammatory effects in post-status epilepticus models of TLE (Gao et al., 2012; Pitsch et al., 2019). The main pharmacologic effect of fingolimod is immunomodulation of lymphocyte homing, thereby reducing the numbers of T and B cells in circulation and, as a consequence, reducing lymphocyte migration into the CNS (Chun et al., 2019). In addition, fingolimod acts on CNS resident cells and inhibits the activation of astrocytes and microglia (Bascunana et al., 2020). Thus, this drug may be an interesting candidate for epilepsy prevention studies in viral encephalitis models, including the TMEV model. However, as shown in Figure 4, epileptogenesis is a complex multifactorial process, so it seems unlikely that affecting neuroinflammation alone will be sufficient to halt this process. We have proposed previously that multi-targeted cocktails of drugs may provide a more effective strategy for epilepsy prevention after brain insults (Löscher, 2021; Löscher and Klein, 2021b). Proof of concept of this strategy has been achieved for the intrahippocampal kainate mouse model of TLE (Schidlitzki et al., 2020; Welzel et al., 2021). However, it remains to be evaluated whether this strategy is also viable for epilepsy developing during viral infections. Robust evidence suggests that ictogenesis during acute viral encephalitis is a complex pathophysiological process that is more than just the sum of parts such as neuroinflammation, invasion of peripheral immune effectors, inflammation- and virus-mediated dysregulation of neural circuitry, and lytic and non-lytic neural cell death. In the absence of complete and global viral eradication, the CNS will always be susceptible to the acutely devastating effects of viral infection. We have provided a broad overview of the manifold viruses that may, directly and indirectly, impact the CNS and we have offered specific mechanistic profiles that offer valuable insights into the potential use of currently available therapeutic strategies to reduce the burden of seizures associated with viral encephalitis. Vitally, we have also offered examples of mechanisms, cell-cell interactions, and host-pathogen interactions that may guide the development of novel therapeutic approaches to preventing, halting, or controlling seizures and the development of epilepsy–not only in patients with viral encephalitis but potentially in many patients with epilepsy and seizure disorders that are refractory to current treatments. Both authors wrote the manuscript and have approved the submission of this work. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. WL’s research described in this review was funded by the Deutsche Forschungsgemeinschaft (Bonn, Germany). CH’s research described in this review was funded by the National Institutes of Health (R01NS064571, R03NS075395, R21NS109956, and R21NS111756) and by the NORSE Institute.

09 5 2022

Frontiers in Molecular Neuroscience

10.3389/fnmol.2022.870868

"Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive (...TRUNCATED)

"Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of e(...TRUNCATED)

"Present address: Aric J. Schultz, Biosciences Center, National Renewable Energy Laboratory, Golden,(...TRUNCATED)

6 8 2019

mBio

10.1128/mBio.01324-19

"An initial exploration of hair cortisol responses to antidepressants\nin unmedicated patients with (...TRUNCATED)

"Major depressive disorder is associated with abnormal functioning of the\nhypothalamic–pituitary(...TRUNCATED)

"These authors contributed equally to this work.\n\nMajor depressive disorder is associated with abn(...TRUNCATED)

18 12 2019

The Journal of International Medical Research

10.1177/0300060519889431

1823. Incidence of Meningoencephalitis in the Absence of CSF Pleocytosis

"Cerebrospinal fluid (CSF) pleocytosis, defined here as ≥5 white blood cells (WBC)/high power fiel(...TRUNCATED)

"Cerebrospinal fluid (CSF) pleocytosis, defined here as ≥5 white blood cells (WBC)/high power fiel(...TRUNCATED)

10 2019

Open Forum Infectious Diseases

10.1093/ofid/ofz359.085

Corrigendum: Clinical spectrum of tauopathies

No abstract available

"Approved by: Frontiers Editorial Office, Frontiers Media SA, Switzerland\n\nThis article was submit(...TRUNCATED)

14 9 2022

Frontiers in Neurology

10.3389/fneur.2022.1015572

Myoclonus in older Cavalier King Charles Spaniels

"Myoclonus is observed in older Cavalier King Charles Spaniels (CKCS) but a full description is lack(...TRUNCATED)

"Myoclonus is observed in older Cavalier King Charles Spaniels (CKCS) but a full description is lack(...TRUNCATED)

23 3 2022

Journal of Veterinary Internal Medicine

10.1111/jvim.16404

Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses

"Human coronaviruses (HCoVs) were first described in the 1960s for patients with the common cold. Si(...TRUNCATED)

"Human coronaviruses (HCoVs) were first described in the 1960s for patients with the common cold. Si(...TRUNCATED)

21 3 2016

Trends in Microbiology

10.1016/j.tim.2016.03.003

Thromboembolic Events in Patients with Influenza: A Scoping Review

"Introduction: Influenza is an acute respiratory infection that usually causes a short-term and self(...TRUNCATED)

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17 12 2022

Viruses

10.3390/v14122817