Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

Analysis of Plasmablasts From Children With Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope.

BACKGROUND: Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. METHODS: We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. RESULTS: We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif. CONCLUSIONS: These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

Fecal microbiome and bile acid metabolome in adult short bowel syndrome.

Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to 2 yr to determine which patients will wean from PN. Here, we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Stool and sera were collected from healthy controls and from patients with SBS (n = 52) with ileostomy, jejunostomy, ileocolonic, and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling, and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS and serum amino acid analyses. Patients with SBS exhibited altered gut microbiota with reduced gut microbial diversity compared with healthy controls. We observed differences in the microbiomes of patients with SBS with ileostomy versus jejunostomy, jejunocolonic versus ileocolonic anastomoses, and PN dependence compared with those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in patients with SBS, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who were weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic aicd. Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select patients with SBS, promoting the ability to wean from PN. Proadaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS.NEW & NOTEWORTHY Loss of intestinal surface area causes short bowel syndrome, intestinal failure, and parenteral nutrition dependence. We analyzed the gut microbiota and bile acid metabolome of a large cohort of short bowel syndrome adult patients with different postsurgical anatomies. We report a novel analysis of the microbiome of patients with ileostomy and jejunostomy. Enrichment of specific microbial and bile acid species may be associated with the ability to wean from parenteral nutrition.

A Protein Epitope Targeted by the Antibody Response to Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1-2 weeks after infection. METHODS: We isolated single peripheral blood plasmablasts from children with KD 1-3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. RESULTS: Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients day ≥ 8 after illness onset, compared with 0/17 infant controls (P < .01), recognized the KD peptide antigen. CONCLUSIONS: These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.

Impaired Chylomicron Assembly Modifies Hepatic Metabolism Through Bile Acid-Dependent and Transmissible Microbial Adaptations.

The mechanisms by which alterations in intestinal bile acid (BA) metabolism improve systemic glucose tolerance and hepatic metabolic homeostasis are incompletely understood. We examined metabolic adaptations in mice with conditional intestinal deletion of the abetalipoproteinemia (ABL) gene microsomal triglyceride transfer protein (Mttp-IKO), which blocks chylomicron assembly and impairs intestinal lipid transport. Mttp-IKO mice exhibit improved hepatic glucose metabolism and augmented insulin signaling, without weight loss. These adaptations included decreased BA excretion, increased pool size, altered BA composition, and increased fibroblast growth factor 15 production. Mttp-IKO mice absorb fructose normally but are protected against dietary fructose-induced hepatic steatosis, without weight loss or changes in energy expenditure. In addition, Mttp-IKO mice exhibit altered cecal microbial communities, both at baseline and following fructose feeding, including increased abundance of Bacteroides and Lactobacillus genera. Transplantation of cecal microbiota from chow-fed Mttp-IKO mice into antibiotic-treated wild-type recipients conferred transmissible protection against fructose-induced hepatic steatosis in association with a bloom in Akkermansia and increased Clostridium XIVa genera, whose abundance was positively correlated with fecal coprostanol and total neutral sterol excretion in recipient mice. However, antibiotic-treated Mttp-IKO mice were still protected against fructose-induced hepatic steatosis, suggesting that changes in microbiota are not required for this phenotype. Nevertheless, we found increased abundance of fecal Akkermansia from two adult ABL subjects with MTTP mutations compared to their heterozygous parents and within the range noted in six healthy control subjects. Furthermore, Akkermansia abundance across all subjects was positively correlated with fecal coprostanol excretion. Conclusion: The findings collectively suggest multiple adaptive pathways of metabolic regulation following blocked chylomicron assembly, including shifts in BA signaling and altered microbial composition that confer a transmissible phenotype.

Enhanced virome sequencing using targeted sequence capture.

Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications.

Detection of Viruses in Clinical Samples by Use of Metagenomic Sequencing and Targeted Sequence Capture.

Metagenomic shotgun sequencing (MSS) is a revolutionary approach to viral diagnostic testing that allows simultaneous detection of a broad range of viruses, detailed taxonomic assignment, and detection of mutations associated with antiviral drug resistance. To enhance sensitivity for virus detection, we previously developed ViroCap, a targeted sequence capture panel designed to enrich nucleic acid from a comprehensive set of eukaryotic viruses prior to sequencing. To demonstrate the utility of MSS with targeted sequence capture for detecting clinically important viruses and characterizing clinically important viral features, we used ViroCap to analyze clinical samples from a diagnostic virology laboratory containing a broad range of medically relevant viruses. From 26 samples, MSS with ViroCap detected all of the expected viruses and 30 additional viruses. Comparing sequencing after capture enrichment with standard MSS, we detected 13 viruses only with capture enrichment and observed a consistent increase in the number and percentage of viral sequence reads as well as the breadth and depth of coverage of the viral genomes. Compared with clinical testing, MSS enhanced taxonomic assignment for 15 viruses, and codons associated with antiviral drug resistance in influenza A virus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV) could be analyzed. Overall, in clinical samples, MSS with targeted sequence capture provides enhanced virus detection and information of clinical and epidemiologic relevance compared with clinical testing and MSS without targeted sequence capture.

BreakPoint Surveyor: a pipeline for structural variant visualization.

SUMMARY: BreakPoint Surveyor (BPS) is a computational pipeline for the discovery, characterization, and visualization of complex genomic rearrangements, such as viral genome integration, in paired-end sequence data. BPS facilitates interpretation of structural variants by merging structural variant breakpoint predictions, gene exon structure, read depth, and RNA-sequencing expression into a single comprehensive figure. AVAILABILITY AND IMPLEMENTATION: Source code and sample data freely available for download at https://github.com/ding-lab/BreakPointSurveyor, distributed under the GNU GPLv3 license, implemented in R, Python and BASH scripts, and supported on Unix/Linux/OS X operating systems. CONTACT: lding@wustl.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The vaginal eukaryotic DNA virome and preterm birth.

BACKGROUND: Despite decades of attempts to link infectious agents to preterm birth, an exact causative microbe or community of microbes remains elusive. Culture-independent sequencing of vaginal bacterial communities demonstrates community characteristics are associated with preterm birth, although none are specific enough to apply clinically. Viruses are important components of the vaginal microbiome and have dynamic relationships with vaginal bacterial communities. We hypothesized that vaginal eukaryotic DNA viral communities (the "vaginal virome") either alone or in the context of bacterial communities are associated with preterm birth. OBJECTIVE: The objective of this study was to use high-throughput sequencing to examine the vaginal eukaryotic DNA virome in a cohort of pregnant women and examine associations between vaginal community characteristics and preterm birth. STUDY DESIGN: This is a nested case-control study within a prospective cohort study of women with singleton pregnancies, not on supplemental progesterone, and without cervical cerclage in situ. Serial midvaginal swabs were obtained at routine prenatal visits. DNA was extracted, bacterial communities were characterized by 16S ribosomal RNA gene sequencing, and eukaryotic viral communities were characterized by enrichment of viral nucleic acid with the ViroCap targeted sequence capture panel followed by nucleic acid sequencing. Viral communities were analyzed according to presence/absence of viruses, diversity, dynamics over time, and association with bacterial community data obtained from the same specimens. RESULTS: Sixty subjects contributed 128 vaginal swabs longitudinally across pregnancy. In all, 24 patients delivered preterm. Participants were predominantly African American (65%). Six families of eukaryotic DNA viruses were detected in the vaginal samples. At least 1 virus was detected in 80% of women. No specific virus or group of viruses was associated with preterm delivery. Higher viral richness was significantly associated with preterm delivery in the full group and in the African American subgroup (P = .0005 and P = .0003, respectively). Having both high bacterial diversity and high viral diversity in the first trimester was associated with the highest risk for preterm birth. CONCLUSION: Higher vaginal viral diversity is associated with preterm birth. Changes in vaginal virome diversity appear similar to changes in the vaginal bacterial microbiome over pregnancy, suggesting that underlying physiology of pregnancy may regulate both bacterial and viral communities.

The lower airway microbiota in early cystic fibrosis lung disease: a longitudinal analysis.

RATIONALE: In infants and young children with cystic fibrosis, lower airway infection and inflammation are associated with adverse respiratory outcomes. However, the role of lower airway microbiota in the pathogenesis of early cystic fibrosis lung disease remains uncertain. OBJECTIVES: To assess the development of the lower airway microbiota over time in infants and young children with cystic fibrosis, and to explore its association with airway inflammation and pulmonary function at age 6 years. METHODS: Serial, semi-annual bronchoscopies and bronchoalveolar lavage (BAL) procedures were performed in infants newly diagnosed with cystic fibrosis following newborn screening. Quantitative microbiological cultures and inflammatory marker (interleukin 8 and neutrophil elastase) measurements were undertaken contemporaneously. 16S ribosomal RNA gene sequencing was conducted on stored BAL samples. Spirometry results recorded at 6 years of age were extracted from medical records. MEASUREMENTS AND MAIN RESULTS: Ninety-five BAL samples provided 16S ribosomal RNA gene data. These were collected from 48 subjects aged 1.2-78.3 months, including longitudinal samples from 27 subjects and 13 before age 6 months. The lower airway microbiota varied, but diversity decreased with advancing age. Detection of recognised cystic fibrosis bacterial pathogens was associated with reduced microbial diversity and greater lower airway inflammation. There was no association between the lower airway microbiota and pulmonary function at age 6 years. CONCLUSIONS: In infants with cystic fibrosis, the lower airway microbiota is dynamic. Dominance of the microbiota by recognised cystic fibrosis bacterial pathogens is associated with increased lower airway inflammation, however early microbial diversity is not associated with pulmonary function at 6 years of age.

Early pregnancy vaginal microbiome trends and preterm birth.

BACKGROUND: Despite decades of attempts to link infectious agents to preterm birth, an exact causative microbe or community of microbes remains elusive. Nonculture 16S ribosomal RNA gene sequencing suggests important racial differences and pregnancy specific changes in the vaginal microbial communities. A recent study examining the association of the vaginal microbiome and preterm birth documented important findings but was performed in a predominantly white cohort. Given the important racial differences in bacterial communities within the vagina as well as persistent racial disparities in preterm birth, it is important to examine cohorts with varied demographic compositions. OBJECTIVE: To characterize vaginal microbial community characteristics in a large, predominantly African-American, longitudinal cohort of pregnant women and test whether particular vaginal microbial community characteristics are associated with the risk for subsequent preterm birth. STUDY DESIGN: This is a nested case-control study within a prospective cohort study of women with singleton pregnancies, not on supplemental progesterone, and without cervical cerclage in situ. Serial mid-vaginal swabs were obtained by speculum exam at their routine prenatal visits. Sequencing of the V1V3 region of the 16S rRNA gene was performed on the Roche 454 platform. Alpha diversity community characteristics including richness, Shannon diversity, and evenness as well as beta diversity metrics including Bray Curtis Dissimilarity and specific taxon abundance were compared longitudinally in women who delivered preterm to those who delivered at term. RESULTS: A total of 77 subjects contributed 149 vaginal swabs longitudinally across pregnancy. Participants were predominantly African-American (69%) and had a preterm birth rate of 31%. In subjects with subsequent term delivery, the vaginal microbiome demonstrated stable community richness and Shannon diversity, whereas subjects with subsequent preterm delivery had significantly decreased vaginal richness, diversity, and evenness during pregnancy (P < .01). This change occurred between the first and second trimesters. Within-subject comparisons across pregnancy showed that preterm birth is associated with increased vaginal microbiome instability compared to term birth. No distinct taxa were associated with preterm birth. CONCLUSION: In a predominantly African-American population, a significant decrease of vaginal microbial community richness and diversity is associated with preterm birth. The timing of this suppression appears early in pregnancy, between the first and second trimesters, suggesting that early gestation may be an ecologically important time for events that ordain subsequent term and preterm birth outcomes.

Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens.

The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for Clostridium difficile identification in diarrhea stool samples. Twenty-two C. difficile-positive diarrhea samples identified by PCR and qPCR and five C. difficile-negative diarrhea controls were studied. C. difficile was detected in 90.9% of C. difficile-positive samples using 16S rRNA gene sequencing, and C. difficile was detected in 86.3% of C. difficile-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of C. difficile from 16S rRNA gene sequencing (r(2) = -0.60) and MSS (r(2) = -0.55). C. difficile was codetected with Clostridium perfringens, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of Candida spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without a priori knowledge.

The transcriptional profile of coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki Disease (KD) can cause potentially life-threatening coronary arteritis in young children, and has a likely infectious etiology. Transcriptome profiling is a powerful approach to investigate gene expression in diseased tissues. RNA sequencing of KD coronary arteries could elucidate the etiology and the host response, with the potential to improve KD diagnosis and/or treatment. METHODS: Deep RNA sequencing was performed on KD (n = 8) and childhood control (n = 7) coronary artery tissues, revealing 1074 differentially expressed mRNAs. Non-human RNA sequences were subjected to a microbial discovery bioinformatics platform, and microbial sequences were analyzed by Metastats for association with KD. RESULTS: T lymphocyte activation, antigen presentation, immunoglobulin production, and type I interferon response were significantly upregulated in KD arteritis, while the tumor necrosis factor α pathway was not differentially expressed. Transcripts from known infectious agents were not specifically associated with KD coronary arteritis. CONCLUSIONS: The immune transcriptional profile in KD coronary artery tissues has features of an antiviral immune response such as activated cytotoxic T lymphocyte and type I interferon-induced gene upregulation. These results provide new insights into the pathogenesis of KD arteritis that can guide selection of new immunomodulatory therapies for high-risk KD patients, and provide direction for future etiologic studies.

Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

Metagenomic analysis of double-stranded DNA viruses in healthy adults.

BACKGROUND: The Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput, metagenomic sequencing analysis. The viruses present in these microbial communities, the 'human virome', are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats. RESULTS: We detected an average of 5.5 viral genera in each individual. At least 1 virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time. CONCLUSIONS: This study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health.

A proautophagic antiviral role for the cellular prion protein identified by infection with a herpes simplex virus 1 ICP34.5 mutant.

The cellular prion protein (PrP) often plays a cytoprotective role by regulating autophagy in response to cell stress. The stress of infection with intracellular pathogens can stimulate autophagy, and autophagic degradation of pathogens can reduce their replication and thus help protect the infected cells. PrP also restricts replication of several viruses, but whether this activity is related to an effect on autophagy is not known. Herpes simplex virus 1 (HSV-1) effectively counteracts autophagy through binding of its ICP34.5 protein to the cellular proautophagy protein beclin-1. Autophagy can reduce replication of an HSV-1 mutant, Δ68H, which is incapable of binding beclin-1. We found that deletion of PrP in mice complements the attenuation of Δ68H, restoring its capacity to replicate in the central nervous system (CNS) to wild-type virus levels after intracranial or corneal infection. Cultured primary astrocytes but not neurons derived from PrP(-/-) mice also complemented the attenuation of Δ68H, enabling Δ68H to replicate at levels equivalent to wild-type virus. Ultrastructural analysis showed that normal astrocytes exhibited an increase in the number of autophagosomes after infection with Δ68H compared with wild-type virus, but PrP(-/-) astrocytes failed to induce autophagy in response to Δ68H infection. Redistribution of EGFP-LC3 into punctae occurred more frequently in normal astrocytes infected with Δ68H than with wild-type virus, but not in PrP(-/-) astrocytes, corroborating the ultrastructural analysis results. Our results demonstrate that PrP is critical for inducing autophagy in astrocytes in response to HSV-1 infection and suggest that PrP positively regulates autophagy in the mouse CNS.

Molecular detection of bacteria, placental inflammation, and neonatal sepsis risk.

OBJECTIVE: To identify bacteria in umbilical cord tissue and investigate the association with placental inflammation and neonatal sepsis risk score. STUDY DESIGN: Retrospective cohort study from 2017-2019. RNA was extracted from umbilical cord tissue and NanoString nCounter used to identify seven bacteria genera. Sepsis risk score was calculated using the Kaiser sepsis calculator. Placental histopathology was abstracted from medical records. RESULTS: Detection of bacterial RNA in the umbilical cord (n = 96/287) was associated with high-stage maternal and fetal acute placental inflammation (maternal 35.4% vs 22.5%, p = 0.03 and fetal 34.4% vs 19.4%, p < 0.01) and maternal vascular malperfusion (36.5% vs 23.0%, p = 0.02). Detection of Ureaplasma spp. was also associated with increased sepsis risk score (1.5/1000 [0.6, 8.6] vs 0.9/1000 [0.2, 2.9], p = 0.04). CONCLUSION: Umbilical cord bacterial pathogens are linked to fetal and maternal placental inflammation and maternal vascular malperfusion during gestation and associated with increased sepsis risk score in the neonate.

The plasma virome in longitudinal samples from pregnant patients.

Introduction: Nucleic acid from viruses is common in peripheral blood, even in asymptomatic individuals. How physiologic changes of pregnancy impact host-virus dynamics for acute, chronic, and latent viral infections is not well described. Previously we found higher viral diversity in the vagina during pregnancy associated with preterm birth (PTB) and Black race. We hypothesized that higher diversity and viral copy numbers in the plasma would show similar trends. Methods: To test this hypothesis, we evaluated longitudinally collected plasma samples from 23 pregnant patients (11 term and 12 preterm) using metagenomic sequencing with ViroCap enrichment to enhance virus detection. Sequence data were analyzed with the ViroMatch pipeline. Results: We detected nucleic acid from at least 1 virus in at least 1 sample from 87% (20/23) of the maternal subjects. The viruses represented 5 families: Herpesviridae, Poxviridae, Papillomaviridae, Anelloviridae, and Flaviviridae. We analyzed cord plasma from 18 of the babies from those patients and found nucleic acid from viruses in 33% of the samples (6/18) from 3 families: Herpesviridae, Papillomaviridae, and Anelloviridae. Some viral genomes were found in both maternal plasma and cord plasma from maternal-fetal pairs (e.g. cytomegalovirus, anellovirus). We found that Black race associated with higher viral richness (number of different viruses detected) in the maternal blood samples (P=0.003), consistent with our previous observations in vaginal samples. We did not detect associations between viral richness and PTB or the trimester of sampling. We then examined anelloviruses, a group of viruses that is ubiquitous and whose viral copy numbers fluctuate with immunological state. We tested anellovirus copy numbers in plasma from 63 pregnant patients sampled longitudinally using qPCR. Black race associated with higher anellovirus positivity (P<0.001) but not copy numbers (P=0.1). Anellovirus positivity and copy numbers were higher in the PTB group compared to the term group (P<0.01, P=0.003, respectively). Interestingly, these features did not occur at the time of delivery but appeared earlier in pregnancy, suggesting that although anelloviruses were biomarkers for PTB they were not triggering parturition. Discussion: These results emphasize the importance of longitudinal sampling and diverse cohorts in studies of virome dynamics during pregnancy.

Virome Analysis and Association of Positive Coxsackievirus B Serology during Pregnancy with Congenital Heart Disease.

BACKGROUND: We have previously shown coxsackievirus B (CVB) to be a potent inducer of congenital heart disease (CHD) in mice. The clinical relevance of these findings in humans and the roles of other viruses in the pathogenesis of CHD remain unknown. METHODS: We obtained plasma samples, collected at all trimesters, from 89 subjects (104 pregnancies), 73 healthy controls (88 pregnancies), and 16 with CHD-affected birth (16 pregnancies), from the Perinatal Family Tissue Bank (PFTB). We performed CVB IgG/IgM serological assays on plasma. We also used ViroCap sequencing and PCR to test for viral nucleic acid in plasma, circulating leukocytes from the buffy coat, and in the media of a co-culture system. RESULTS: CVB IgG/IgM results indicated that prior exposure was 7.8 times more common in the CHD group (95% CI, 1.14-54.24, adj. p-value = 0.036). However, the CVB viral genome was not detected in plasma, buffy coat, or co-culture supernatant by molecular assays, although other viruses were detected. CONCLUSION: Detection of viral nucleic acid in plasma was infrequent and specifically no CVB genome was detected. However, serology demonstrated that prior CVB exposure is higher in CHD-affected pregnancies. Further studies are warranted to understand the magnitude of the contribution of the maternal blood virome to the pathogenesis of CHD.

Metagenomic Analysis of DNA Viruses with Targeted Sequence Capture of Canine Lobular Orbital Adenomas and Normal Conjunctiva.

Our study aims are: (1) to evaluate phenotypically normal canine conjunctival and orbital tissue and tissue from canine lobular orbital adenomas (CLOAs) for the presence of viral genomic material and (2) phylogenetically classify detected DNA viruses to determine if a DNA virus is associated with CLOAs. A total of 31 formalin fixed paraffin embedded CLOA tissue samples, 4 papillomas or sarcoid, and 10 fresh clinically normal conjunctival tissues were included in this study. Genomic DNA was isolated from all samples and sequencing libraries were prepared. The libraries were molecularly indexed and pooled and viral DNA was enriched via targeted sequence capture utilizing ViroCap. The libraries were sequenced on the Illumina HiSeq platform and compared to known viral DNA reference genomes to identify viral DNA. Carnivore parvovirus was identified in 6.4% and 20% of CLOA tissue and normal conjunctival samples, respectively. This study showed that conjunctival tissue from healthy dogs and CLOAs uncommonly harbor DNA viruses, and no DNA virus was associated with these tumors. Further studies are needed to evaluate the etiologic cause of CLOAs.