Human cytomegalovirus (HCMV) is the most common viral infection acquired in utero and a leading cause of neurodevelopmental abnormalities, including sensorineural hearing loss (SNHL). In previous studies using a murine model of HCMV induced SNHL, hearing loss was correlated with virus-induced cochlear inflammation but not cochlear viral load. However, these previous findings were determined at the time of auditory testing, a time poiont well past critical periods of auditory development. In the current study, cochlear virus load early in auditory development could be correlated with the magnitude of virus-induced cochlear inflammation, cochlear histopathology and the development of hearing loss. Transcriptional profiling at early times after infection revealed dysregulation of multiple well described deafness-related genes (DRG). Treatment with antiviral antibodies early after infection decreased cochlear virus load, cochlear inflammation, cochlear histopathology, and normalized DRG expression arguing that virus-induced cochlear inflammation can result in pleiotropic effects on the developing auditory system. Finally, this model also demonstrated that sterilizing immunity was unnecessary for prevention of SNHL, thus providing a rationale for inteventions that could limit, but not completely prevent HCMV infection of the developing auditory system.

Abstract Background The majority of COVID-19 infections in children, including in those with underlying hematological and oncological (Heme-Onc) diagnoses are mild with good outcome compared to adults. Postulated reasons include cross-protection from immunity against seasonal coronaviruses (HCoV’s), subdued inflammatory responses in children than in adults and differences in humoral and T-cell responses. The objectives of this study are to compare antibody responses against HCoV’s, anti-SARS-CoV-2 humoral, T-cell, and inflammatory profiles between Heme-Onc children with COVID-19 and control groups. Methods Binding IgG antibodies against Spike (S) of SARS-CoV-2, four HCoV’s (HKU1, OC43, NL63 and 229E), quantitative levels of 21 cytokines and 9 chemokines (multiplexed solid-phase electrochemiluminescence assay by Meso Scale Diagnostics/MSD) were compared among five cohorts: 1) children with Heme-Onc diagnoses and COVID-19 (n = 41) 2) healthy children with COVID-19 (n = 21) 3) adults with COVID-19 (n = 14) 4) children with multisystem inflammatory syndrome (MIS-C) [n = 23] and 5) healthy children without COVID-19 (n = 12). Additionally, T-cell responses against S and nucleocapsid (N) proteins of SARS-CoV-2 was assessed using ELISpot in Heme-Onc children a group of healthy children COVID-19. Results Anti-S antibodies and neutralizing antibodies against SARS-CoV-2 and HCoV were significantly lower in children in Heme-Onc and healthy children with COVID-19 compared to adults with COVID-19 (Figure 1). The concentration of eight pro-inflammatory cytokines and chemokines were significantly lower in Heme-Onc and healthy children compared to adults with COVID-19 and children with MIS-C. The responder T-cell frequency varied between 0 and 860/100,000 PBMC at study enrollment in children with Heme-Onc diagnoses. Conclusion In this single-center study, cross-protection from HCoV antibodies is an unlikely mechanism to explain mild COVID-19 in children, including in those with underlying hematological and oncological diagnoses. Subdued inflammatory responses to SARS-CoV-2 could be one of the factors for the mild COVID-19 disease phenotype in children. Disclosures Swetha Pinninti, MD, Moderna: Grant/Research Support|Pfizer: Grant/Research Support Suresh Boppana, MD, GSK: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Grant/Research Support

Before Zika virus (ZIKV) infections were observed in the Americas, an association between ZIKV and microcephaly or other congenital malformations was not well documented. Initial reports suggested strong associations between ZIKV and congenital malformations, but plausible estimates of causal effects from prospective studies with adequate sample size and covariate data were few. From 2016–2018, the Zika in Infants and Pregnancy (ZIP) study enrolled pregnant people before 18 weeks gestation or with confirmed symptomatic ZIKV in a prospective cohort across 10 sites in South and Central America, and in Puerto Rico. Pregnancies were followed monthly through delivery and 6 weeks postpartum. Infants were followed quarterly to age 12 months. Prespecified co-primary analyses evaluated the associations between a composite endpoint of adverse fetal, neonatal, and infant outcomes with intrauterine ZIKV exposure overall and with symptomatic intrauterine ZIKV exposure. Secondary analyses separately evaluated the association of intrauterine ZIKV exposure with individual components of the primary endpoint. Six thousand one hundred pregnant participants were included in the primary analysis, including 61 with ZIKV infection during pregnancy confirmed by a ZIKV-specific RNA test. For the primary analyses, the relative risk (RR) for the composite endpoint associated with any ZIKV exposure was 1.64 (95

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Human cytomegalovirus (HCMV) infection in infants infected in utero can lead to a variety of neurodevelopmental disorders. However, mechanisms underlying altered neurodevelopment in infected infants remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in all regions of the brain of newborn mice, including the cerebellum. Infection resulted in disruption of cerebellar cortical development characterized by reduced cerebellar size and foliation. This disruption was associated with altered cell cycle progression of the granule cell precursors (GCPs), which are the progenitors that give rise to granule cells (GCs), the most abundant neurons in the cerebellum. In the current study, we have demonstrated that MCMV infection leads to prolonged GCP cell cycle, premature exit from the cell cycle, and reduced numbers of GCs resulting in cerebellar hypoplasia. Treatment with TNF-α neutralizing antibody partially normalized the cell cycle alterations of GCPs and altered cerebellar morphogenesis induced by MCMV infection. Collectively, our results argue that virus-induced inflammation altered the cell cycle of GCPs resulting in a reduced numbers of GCs and cerebellar cortical hypoplasia, thus providing a potential mechanism for altered neurodevelopment in fetuses infected with HCMV.

Human cytomegalovirus is a medically important pathogen. Previously, using murine CMV (MCMV), we provided evidence that both neutralizing and nonneutralizing antibodies can confer protection from viral infection in vivo. In this study, we report that serum derived from infected animals had a greater protective capacity in MCMV-infected RAG-/- mice than serum from animals immunized with purified virus. The protective activity of immune serum was strictly dependent on functional Fcγ receptors (FcγR). Deletion of individual FcγRs or combined deletion of FcγRI and FcγRIV had little impact on the protection afforded by serum. Adoptive transfer of CD115-positive cells from noninfected donors demonstrated that monocytes represent important cellular mediators of the protective activity provided by immune serum. Our studies suggest that Fc-FcγR interactions and monocytic cells are critical for antibody-mediated protection against MCMV infection in vivo. These findings may provide new avenues for the development of novel strategies for more effective CMV vaccines or antiviral immunotherapies.

Congenital cytomegalovirus infection (cCMV) is a frequent cause of non-hereditary sensorineural hearing loss (SNHL) and developmental disabilities. The contribution of cCMV to childhood hearing loss has been estimated to be about 25% of all hearing loss in children at 4 years of age. Although the vestibular insufficiency (VI) in cCMV has not been well-characterized and therefore, underestimated, recent studies suggest that VI is also frequent in children with cCMV and can lead to adverse neurodevelopmental outcomes. The pathogenesis of SNHL and VI in children with cCMV has been thought to be from direct viral cytopathic effects as well as local inflammatory responses playing a role. Hearing loss in cCMV can be of varying degrees of severity, unilateral or bilateral, present at birth or develop later (late-onset), and can progress or fluctuate in early childhood. Therefore, newborn hearing screening fails to identify a significant number of children with CMV-related SNHL. Although the natural history of cCMV-associated VI has not been well characterized, recent data suggests that it is likely that VI also varies considerably with respect to the laterality, timing of onset, degree of the deficit, and continued deterioration during early childhood. This article summarizes the current understanding of the natural history and pathogenesis of auditory and vestibular disorders in children with cCMV.

Microglia are myeloid cells that reside within the central nervous system (CNS), where they maintain homeostasis under normal, non-pathological conditions. In addition, microglia also perform numerous immune functions upon different pathogenic stimuli, including CNS infections with various neurotropic viruses. Herpesviruses establish a lifelong latent infection from which they reactivate intermittently upon waning of immune control. The role of microglia in preventing reactivation of latent herpesviruses remains unclear. In this work, we used congenital cytomegalovirus (CMV) infection as a model to investigate the impact of a persistent virus infection of the brain on microglia. We show that mouse CMV (MCMV) latency in the CNS is associated with permanent microglial priming. The changes induced by persistent infection include continuous, interferon-gamma-dependent microglia activation and extensive transcriptional reprogramming at the single-cell level, leading to the expansion of a microglia subset associated with latent infection. Notably, the maintenance of microglia in a primed state provides enhanced control of latent infection and superior recall response but is associated with excessive loss of synaptic dendritic spines mediated by primed microglia. Altogether, our results indicate that latent CMV infection in the brain causes perturbation of microglial homeostasis, which leads to chronic neuroinflammation that successfully restricts virus reactivation but simultaneously compromises neuronal synaptic connectivity in the brain.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA 1 , 2 . Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA 3 – 7 , although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls ( P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology ( P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases ( P < 0.001). Co-infections by Epstein–Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls ( P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike’s fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike’s fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

Congenital Human Cytomegalovirus (HCMV) infection is a leading cause of sensorineural hearing loss (SNHL) in children. The mechanism of HCMV damage to the developing auditory system that leads to SNHL is not understood and effective treatments are lacking. We developed a murine model of HCMV infection that results in neurodevelopmental abnormalities and SNHL, thus providing a useful model to study the mechanisms of SNHL associated with cCMV infection. We have shown that MCMV induces expression of proinflammatory molecules and immune cell activation in the cochlea associated with histopathologic changes and SNHL. The stria vascularis (SV) plays a critical role in hearing by; (1) maintaining an epithelial barrier needed for generation of the high electrical potential of endocochlear fluid surrounding the hair cells; (2) expression of a system of ion channels that create a high K +concentration in the endocochlear fluid; and (3) generation of a blood labyrinth barrier (BLB) that surrounds the vasculature in the SV and functions like the blood brain barrier. Utilizing several assays, our studies showed that MCMV infection altered each of these functions. The transcription and protein expression in the SV required for tight junctions in the epithelial barrier and ion channels were decreased early after infection and in SV from infected mice with SNHL compared to those without SNHL. In addition, the resident macrophages that contribute to the BLB had altered phenotypes. Functionally, penetration of a cell-impermeable tracer into the SV could be demonstrated in MCMV infected mice. In all, our data show that MCMV dysregulates expression of key proteins in the SV that in turn impact the capacity of the SV to maintain the cochlea’s unique environment.

Reactivation of human cytomegalovirus (HCMV) from latency is a frequent complication following hematopoietic stem cell transplantation (HSCT). The development of acute graft-versus-host disease (GVHD) is a significant risk factor for HCMV disease. Using a murine GVHD model in animals latently infected with murine CMV (MCMV), we studied preventive and therapeutic interventions in this high-risk scenario of HSCT. Mice latently infected with MCMV experienced reactivated MCMV and developed disseminated MCMV infection concomitant with the manifestations of GVHD. Dissemination was accompanied by accelerated mortality. We demonstrate that MCMV reactivation and dissemination was modulated by MCMV-specific antibodies, thus demonstrating in vivo protective activity of antiviral antibodies. However, the efficacy of serum therapy required repetitive doses of high-titer immune serum secondary to the shortened serum half-life of IgG in animals with GVHD. In a complementary approach, treatment of GVHD by adoptive transfer of donor-derived Tregs facilitated production of MCMV-specific antibodies from newly developing donor-derived B cells. Together, our findings strongly suggest that antibodies play a major role in controlling recurrent MCMV infection that follows GVHD, and they argue for reassessing the potential of antibody treatments as well as therapeutic strategies that enhance de novo antibody development against HCMV.

Severe acute hepatitis of unknown etiology in children is under investigation in 35 countries. Although several potential etiologic agents have been investigated, a clear cause for the liver damage observed in these cases remains to be identified. Using VirScan, a high-throughput antibody profiling technology, we probed the antibody repertoires of nine cases of severe acute hepatitis of unknown etiology treated at Children's of Alabama and compared their antibody responses with 38 pediatric and 470 adult controls. We report increased adeno-associated dependoparvovirus A (AAV-A) breadth in cases relative to controls and adeno-associated virus 2 (AAV-2) peptide responses that were conserved in seven of nine cases but rarely observed in pediatric and adult controls. These findings suggest that AAV-2 is a likely etiologic agent of severe acute hepatitis of unknown etiology.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.

Abstract Background Human adenoviruses (HAdV) are a common cause of respiratory and gastrointestinal infections but rarely cause end-organ disease in children. From October 2021 to February 2022, several previously healthy children admitted to a single center for significant hepatitis also tested positive for HAdV. The aim of this investigation is to describe characteristics of these children. Methods Children admitted to Children’s of Alabama (COA) from October 2021 to February 2022 with hepatitis who tested positive for HAdV by whole blood RT-PCR were included. Demographic, clinical, laboratory and treatment data were collected from medical records. Residual blood specimens were sent for adenovirus typing. Results Nine pediatric patients with hepatitis and HAdV infection were identified (78% female; median age 3.0 years; IQR 1.7-3.0). Before admission, six reported diarrhea and three had respiratory symptoms. At presentation, eight had scleral icterus, six had jaundice, seven had hepatomegaly, and one was encephalopathic. All patients had elevated transaminases (AST range: 447-4000 U/L, ALT range: 784-4695 U/L); initial total bilirubin varied [range 0.23-13.5 mg/dL]). All had confirmed HAdV by RT-PCR on whole blood (initial qPCR range: 991-70,680 copies/mL). Notably, two children who transferred to another facility were negative for HAdV by RT-PCR when plasma was tested (instead of whole blood). Six children underwent liver biopsy showing varying degrees of hepatitis with no adenovirus detected on immunohistochemistry stains. Five children had HAdV type 41 confirmed. Three children presented or progressed to acute liver failure, two children were treated with cidofovir, and two underwent successful liver transplantation. No known epidemiologic links between patients were identified and all were from geographically distinct parts of Alabama. Conclusion HAdV is a potentially underrecognized cause of hepatitis. Whole blood specimens may be preferred over plasma for HAdV RT-PCR testing. HAdV type 41 was identified in all patients with typing results available. Improved type-based surveillance may help determine HAdV patterns of circulation and inform future diagnostic testing. Disclosures William Britt, MD, First Energy Corporation: Stocks/Bonds|Hookipa Pharma: Advisor/Consultant|Kroger Care: Stocks/Bonds|MDU Resources Group: Stocks/Bonds|PG&E Corporation: Stocks/Bonds.

Human cytomegalovirus (HCMV) infection of the developing central nervous system (CNS) in infants infected in utero can lead to a variety of neurodevelopmental disorders. Although the link between HCMV infection and neurodevelopmental deficits is widely recognized, underlying mechanisms leading to altered neurodevelopment remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in the brain of newborn mice. Infection results in the disruption of cerebellar cortical development characterized by reduced cerebellar size, but paradoxically, an increase in the number of cerebellar granule cell precursors (GCPs) in the external granular layer (EGL) of the cerebellar cortex. This increased number of GCPs in the EGL is associated with abnormal cell cycle progression and decreased GCP migration from EGL and IGL. In the current study, we demonstrated that MCMV infection led to prolonged G1- and S-phases of the GCP cell cycle and increased cell cycle exit. Treatment with TNFα neutralizing antibody partially normalized the cell cycle progression of GCPs. Collectively, our results argue that inflammation can alter GCP proliferation and lead to premature exit from the cell cycle resulting in reduced cerebellar size in MCMV-infected mice. These findings provide insight into mechanisms of altered brain development of fetuses infected with HCMV and possibly, other infectious agents that induce inflammation during neurodevelopment.

During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood.

Human cytomegalovirus (HCMV) infection of the developing central nervous system (CNS) in infants infected in utero can lead to a variety of neurodevelopmental disorders. Although the link between HCMV infection and neurodevelopmental deficits is widely recognized, underlying mechanisms leading to altered neurodevelopment remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in the brain of newborn mice. Infection results in the disruption of cerebellar cortical development characterized by reduced cerebellar size, but paradoxically, an increase in the number of cerebellar granule cell precursors (GCPs) in the external granular layer (EGL) of the cerebellar cortex. This increased number of GCPs in the EGL is associated with abnormal cell cycle progression and decreased GCP migration from EGL and IGL. In the current study, we demonstrated that MCMV infection led to prolonged G1- and S-phases of the GCP cell cycle and increased cell cycle exit. Treatment with TNFα neutralizing antibody partially normalized the cell cycle progression of GCPs. Collectively, our results argue that inflammation can alter GCP proliferation and lead to premature exit from the cell cycle resulting in reduced cerebellar size in MCMV-infected mice. These findings provide insight into mechanisms of altered brain development of fetuses infected with HCMV and possibly, other infectious agents that induce inflammation during neurodevelopment. ### Competing Interest Statement The authors have declared no competing interest.