In 1983, reports of antibodies in subjects with major depressive disorder to an as-yet uncharacterized infectious agent associated with meningoencephalitis in horses and sheep led to the molecular cloning of the genome of a novel, negative-stranded neurotropic virus, Borna disease virus (BDV).1,2 This advance enabled the development of new diagnostic assays including in situ hybridization, PCR and serology based on recombinant proteins. Since these assays were first implemented in 1990 more than 80 studies have reported an association between BDV and a wide range of human illnesses that include major depressive disorder, bipolar disorder, schizophrenia, anxiety disorder, chronic fatigue syndrome, multiple sclerosis, amyotrophic lateral sclerosis, dementia and glioblastoma multiforme.3,4 However, to date there has been no blinded case-control study of the epidemiology of BDV infection. Here, in a United States-based, multi-center, yoked case-control study with standardized methods for clinical assessment and blinded serologic and molecular analysis, we report the absence of association of psychiatric illness with antibodies to BDV or with BDV nucleic acids in serially-collected serum and white blood cell samples from 396 subjects, a study population comprised of 198 matched pairs of patients and healthy controls (52 schizophrenia/control pairs, 66 bipolar disorder/control pairs, and 80 major depressive disorder/control pairs). Our results argue strongly against a role for BDV in the pathogenesis of these psychiatric disorders.
Borna disease virus; infection; schizophrenia; affective disorders; pathogenesis
The disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (CFS/ME) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (pMLV). Although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. Here we report blinded analysis of peripheral blood from a rigorously characterized, geographically diverse population of 147 patients with CFS/ME and 146 healthy subjects by the investigators describing the original association. This analysis reveals no evidence of either XMRV or pMLV infection.
Chronic fatigue syndrome/myalgic encephalomyelitis has an estimated prevalence of 42/10,000 in the United States, with annual direct medical costs of $7 billion. Here, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with this disorder report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects. The increasing frequency with which molecular methods are used for pathogen discovery poses new challenges to public health and support of science. It is imperative that strategies be developed to rapidly and coherently address discoveries so that they can be carried forward for translation to clinical medicine or abandoned to focus resource investment more productively. Our study provides a paradigm for pathogen dediscovery that may be helpful to others working in this field.
Although autism is first and foremost a disorder of the central nervous system, comorbid dysfunction of the gastrointestinal (GI) and immune systems is common, suggesting that all three systems may be affected by common molecular mechanisms. Substantial systemic deficits in the antioxidant glutathione and its precursor, cysteine, have been documented in autism in association with oxidative stress and impaired methylation. DNA and histone methylation provide epigenetic regulation of gene expression during prenatal and postnatal development. Prenatal epigenetic programming (PrEP) can be affected by the maternal metabolic and nutritional environment, whereas postnatal epigenetic programming (PEP) importantly depends upon nutritional support provided through the GI tract. Cysteine absorption from the GI tract is a crucial determinant of antioxidant capacity, and systemic deficits of glutathione and cysteine in autism are likely to reflect impaired cysteine absorption. Excitatory amino acid transporter 3 (EAAT3) provides cysteine uptake for GI epithelial, neuronal, and immune cells, and its activity is decreased during oxidative stress. Based upon these observations, we propose that neurodevelopmental, GI, and immune aspects of autism each reflect manifestations of inadequate antioxidant capacity, secondary to impaired cysteine uptake by the GI tract. Genetic and environmental factors that adversely affect antioxidant capacity can disrupt PrEP and/or PEP, increasing vulnerability to autism.
Gastrointestinal disturbances are commonly reported in children with autism and may be associated with compositional changes in intestinal bacteria. In a previous report, we surveyed intestinal microbiota in ileal and cecal biopsy samples from children with autism and gastrointestinal dysfunction (AUT-GI) and children with only gastrointestinal dysfunction (Control-GI). Our results demonstrated the presence of members of the family Alcaligenaceae in some AUT-GI children, while no Control-GI children had Alcaligenaceae sequences. Here we demonstrate that increased levels of Alcaligenaceae in intestinal biopsy samples from AUT-GI children result from the presence of high levels of members of the genus Sutterella. We also report the first Sutterella-specific PCR assays for detecting, quantitating, and genotyping Sutterella species in biological and environmental samples. Sutterella 16S rRNA gene sequences were found in 12 of 23 AUT-GI children but in none of 9 Control-GI children. Phylogenetic analysis revealed a predominance of either Sutterella wadsworthensis or Sutterella stercoricanis in 11 of the individual Sutterella-positive AUT-GI patients; in one AUT-GI patient, Sutterella sequences were obtained that could not be given a species-level classification based on the 16S rRNA gene sequences of known Sutterella isolates. Western immunoblots revealed plasma IgG or IgM antibody reactivity to Sutterella wadsworthensis antigens in 11 AUT-GI patients, 8 of whom were also PCR positive, indicating the presence of an immune response to Sutterella in some children.
Autism spectrum disorders affect ~1% of the population. Many children with autism have gastrointestinal (GI) disturbances that can complicate clinical management and contribute to behavioral problems. Understanding the molecular and microbial underpinnings of these GI issues is of paramount importance for elucidating pathogenesis, rendering diagnosis, and administering informed treatment. Here we describe an association between high levels of intestinal, mucoepithelial-associated Sutterella species and GI disturbances in children with autism. These findings elevate this little-recognized bacterium to the forefront by demonstrating that Sutterella is a major component of the microbiota in over half of children with autism and gastrointestinal dysfunction (AUT-GI) and is absent in children with only gastrointestinal dysfunction (Control-GI) evaluated in this study. Furthermore, these findings bring into question the role Sutterella plays in the human microbiota in health and disease. With the Sutterella-specific molecular assays described here, some of these questions can begin to be addressed.
Toll-like receptor 3 (TLR3) signaling has been implicated in neural stem/precursor cell (NPC) proliferation. However, the molecular mechanisms involved, and their relationship to classical TLR-mediated innate immune pathways, remain unknown. Here, we report investigation of the mechanics of TLR3 signaling in neurospheres comprised of epidermal growth factor (EGF)-responsive NPC isolated from murine embryonic cerebral cortex of C57BL/6 (WT) or TLR3 deficient (TLR3−/−) mice. Our data indicate that the TLR3 ligand polyinosinic-polycytidylic acid (PIC) negatively regulates NPC proliferation by inhibiting Sonic Hedgehog (Shh) signaling, that PIC induces apoptosis in association with inhibition of Ras-ERK signaling and elevated expression of Fas, and that these effects are TLR3-dependent, suggesting convergent signaling between the Shh and TLR3 pathways.
Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.
Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.
The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.
Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus  in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses.
HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.
The reported prevalence of autism spectrum disorders (ASD) has increased 5–10× over the past 20 years. Whether ASD are truly more frequent is controversial; nonetheless, the burden is profound in human and economic terms. Although autism is among the most heritable of mental disorders, its pathogenesis remains obscure. Environmental factors are proposed; however, none is implicated. Furthermore, there are no biomarkers to screen for ASD or risk of ASD. The Autism Birth Cohort (ABC) was initiated to investigate gene × environment × timing interactions and enable early diagnosis. It employs a large, unselected birth cohort wherein cases are prospectively ascertained through population screening. Samples collected serially through pregnancy and childhood include parental blood, maternal urine, cord blood, milk teeth and rectal swabs. More than 107 000 children are continuously screened via questionnaires, referral and a national registry. Cases are compared with a control group from the same cohort in a “nested case-control” design. Early screening, diagnostic assessments and re-assessments are designed to provide a rich view of longitudinal trajectory. Genetic, proteomic, immunologic, metagenomic and microbiological tools will be used to exploit unique biological samples. The ABC is a paradigm for investigating the role of genetic and environmental factors in complex disorders.
Autism; neurodevelopmental disorder; birth cohort; biobank; molecular biology; genes and environment
Maternal infection during pregnancy with a wide range of RNA and DNA viruses is associated with increased risk for schizophrenia and autism in their offspring. A common feature in these exposures is that virus replication induces innate immunity through interaction with Toll-like receptors (TLRs). We employed a mouse model wherein pregnant mice were exposed to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA molecular mimic of replicating virus. Poly(I ⋅ C) inhibited embryonic neuronal stem cell replication and population of the superficial layers of the neocortex by neurons. Poly(I ⋅ C) also led to impaired neonatal locomotor development and abnormal sensorimotor gating responses in adult offspring. Using Toll-like receptor 3 (TLR3)-deficient mice, we established that these effects were dependent on TLR3. Inhibition of stem cell proliferation was also abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen, a cyclooxygenase (COX) inhibitor. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and behavioral dysfunction, and they may suggest strategies for reducing the risk of neuropsychiatric disorders subsequent to prenatal exposures to pathogens and other triggers of innate immunity.
Maternal infection during gestation increases the risk of neuropsychiatric disorders in their offspring. Furthermore, work in animal models indicates that pre- or neonatal infections with a wide range of viruses results in similar neurodevelopmental outcomes. These observations are consistent with a mechanism whereby damage is mediated through common pathways. Exposure of pregnant mice to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA (dsRNA) molecular mimic of replicating virus, inhibited embryonic neuronal stem cell replication and led to behavioral abnormalities in their offspring. These effects were mediated through TLR3 and abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and may suggest strategies for reducing the risk of neuropsychiatric diseases following exposures to infectious agents and other triggers of innate immunity during gestation.
Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999, HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.
The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied.
Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful.
The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.
In October 2007, a cluster of patients experiencing a novel polyradiculoneuropathy was identified at a pork abattoir (Plant A). Patients worked in the primary carcass processing area (warm room); the majority processed severed heads (head-table). An investigation was initiated to determine risk factors for illness.
Methods and Results
Symptoms of the reported patients were unlike previously described occupational associated illnesses. A case-control study was conducted at Plant A. A case was defined as evidence of symptoms of peripheral neuropathy and compatible electrodiagnostic testing in a pork abattoir worker. Two control groups were used - randomly selected non-ill warm-room workers (n = 49), and all non-ill head-table workers (n = 56). Consenting cases and controls were interviewed and blood and throat swabs were collected. The 26 largest U.S. pork abattoirs were surveyed to identify additional cases. Fifteen cases were identified at Plant A; illness onsets occurred during May 2004–November 2007. Median age was 32 years (range, 21–55 years). Cases were more likely than warm-room controls to have ever worked at the head-table (adjusted odds ratio [AOR], 6.6; 95% confidence interval [CI], 1.6–26.7), removed brains or removed muscle from the backs of heads (AOR, 10.3; 95% CI, 1.5–68.5), and worked within 0–10 feet of the brain removal operation (AOR, 9.9; 95% CI, 1.2–80.0). Associations remained when comparing head-table cases and head-table controls. Workers removed brains by using compressed air that liquefied brain and generated aerosolized droplets, exposing themselves and nearby workers. Eight additional cases were identified in the only two other abattoirs using this technique. The three abattoirs that used this technique have stopped brain removal, and no new cases have been reported after 24 months of follow up. Cases compared to controls had higher median interferon-gamma (IFNγ) levels (21.7 pg/ml; vs 14.8 pg/ml, P<0.001).
This novel polyradiculoneuropathy was associated with removing porcine brains with compressed air. An autoimmune mechanism is supported by higher levels of IFNγ in cases than in controls consistent with other immune mediated illnesses occurring in association with neural tissue exposure. Abattoirs should not use compressed air to remove brains and should avoid procedures that aerosolize CNS tissue. This outbreak highlights the potential for respiratory or mucosal exposure to cause an immune-mediated illness in an occupational setting.
Initial reports in May 2009 of the novel influenza strain H1N1pdm estimated a case fatality rate (CFR) of 0.6%, similar to that of seasonal influenza. In July 2009, however, Argentina reported 3056 cases with 137 deaths, representing a CFR of 4.5%. Potential explanations for increased CFR included virus reassortment or genetic drift, or infection of a more vulnerable population. Virus genomic sequencing of 26 Argentinian samples representing both severe and mild disease indicated no evidence of reassortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence. Furthermore, no evidence was found for increased frequency of risk factors for H1N1pdm disease.
We examined nasopharyngeal swab samples (NPS) from 199 cases of H1N1pdm infection from Argentina with MassTag PCR, testing for 33 additional microbial agents. The study population consisted of 199 H1N1pdm-infected subjects sampled between 23 June and 4 July 2009. Thirty-nine had severe disease defined as death (n = 20) or hospitalization (n = 19); 160 had mild disease. At least one additional agent of potential pathogenic importance was identified in 152 samples (76%), including Streptococcus pneumoniae (n = 62); Haemophilus influenzae (n = 104); human respiratory syncytial virus A (n = 11) and B (n = 1); human rhinovirus A (n = 1) and B (n = 4); human coronaviruses 229E (n = 1) and OC43 (n = 2); Klebsiella pneumoniae (n = 2); Acinetobacter baumannii (n = 2); Serratia marcescens (n = 1); and Staphylococcus aureus (n = 35) and methicillin-resistant S. aureus (MRSA, n = 6). The presence of S. pneumoniae was strongly correlated with severe disease. S. pneumoniae was present in 56.4% of severe cases versus 25% of mild cases; more than one-third of H1N1pdm NPS with S. pneumoniae were from subjects with severe disease (22 of 62 S. pneumoniae-positive NPS, p = 0.0004). In subjects 6 to 55 years of age, the adjusted odds ratio (OR) of severe disease in the presence of S. pneumoniae was 125.5 (95% confidence interval [CI], 16.95, 928.72; p<0.0001).
The association of S. pneumoniae with morbidity and mortality is established in the current and previous influenza pandemics. However, this study is the first to demonstrate the prognostic significance of non-invasive antemortem diagnosis of S. pneumoniae infection and may provide insights into clinical management.
Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.
Borna disease virus; birds; proventricular dilatation disease; real time PCR; high throughput pyrosequencing; dispatch
The presence of measles virus (MV) RNA in bowel tissue from children with autism spectrum disorders (ASD) and gastrointestinal (GI) disturbances was reported in 1998. Subsequent investigations found no associations between MV exposure and ASD but did not test for the presence of MV RNA in bowel or focus on children with ASD and GI disturbances. Failure to replicate the original study design may contribute to continued public concern with respect to the safety of the measles, mumps, and rubella (MMR) vaccine.
The objective of this case-control study was to determine whether children with GI disturbances and autism are more likely than children with GI disturbances alone to have MV RNA and/or inflammation in bowel tissues and if autism and/or GI episode onset relate temporally to receipt of MMR. The sample was an age-matched group of US children undergoing clinically-indicated ileocolonoscopy. Ileal and cecal tissues from 25 children with autism and GI disturbances and 13 children with GI disturbances alone (controls) were evaluated by real-time reverse transcription (RT)-PCR for presence of MV RNA in three laboratories blinded to diagnosis, including one wherein the original findings suggesting a link between MV and ASD were reported. The temporal order of onset of GI episodes and autism relative to timing of MMR administration was examined. We found no differences between case and control groups in the presence of MV RNA in ileum and cecum. Results were consistent across the three laboratory sites. GI symptom and autism onset were unrelated to MMR timing. Eighty-eight percent of ASD cases had behavioral regression.
This study provides strong evidence against association of autism with persistent MV RNA in the GI tract or MMR exposure. Autism with GI disturbances is associated with elevated rates of regression in language or other skills and may represent an endophenotype distinct from other ASD.
Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.
Infection of newborn Lewis rats with Borna disease virus (neonatal Borna disease [NBD]) results in cerebellar damage without the cellular inflammation associated with infections in later life. Purkinje cell (PC) damage has been reported for several models of early-life viral infection, including NBD; however, the time course and distribution of PC pathology have not been investigated rigorously. This study examined the spatiotemporal relationship between PC death and zonal organization in NBD cerebella. Real-time PCR at postnatal day 28 (PND28) revealed decreased cerebellar levels of mRNAs encoding the glycolytic enzymes aldolase C (AldoC, also known as zebrin II) and phosphofructokinase C and the excitatory amino acid transporter 4 (EAAT4). Zebrin II and EAAT4 immunofluorescence analysis in PND21, PND28, PND42, and PND84 NBD rat cerebella revealed a complex pattern of PC degeneration. Early cell loss (PND28) was characterized by preferential apoptotic loss of zebrin II/EAAT4-negative PC subsets in the anterior vermis. Consistent with early preferential loss of zebrin II/EAAT4-negative PCs in the vermis, the densities of microglia and the Bergmann glial expression of metallothionein I/II and the hyaluronan receptor CD44 were higher in zebrin II/EAAT4-negative zones. In contrast, early loss in lateral cerebellar lobules did not reflect a similar discrimination between PC phenotypes. Patterns of vermal PC loss became more heterogeneous at PND42, with the loss of both zebrin II/EAAT4-negative and zebrin II/EAAT4-positive neurons. At PND84, zebrin II/EAAT4 patterning was abolished in the anterior cerebellum, with preferential PC survival in lobule X. Our investigation reveals regional discrimination between patterns of PC subset loss, defined by zebrin II/EAAT4 expression domains, following neonatal viral infection. These findings suggest a differential vulnerability of PC subsets during the early stages of virus-induced neurodegeneration.