Molecular profiling of the hippocampus of children with autism spectrum disorder.

Several lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n = 7) and normal male children (3-14 yrs old), (n = 6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p < 0.02) and SYN1 (p < 0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p < 0.03), and decreased expression of MEF2C (p < 0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.

Sex and molecular differences in cardiovascular parameters at peak influenza disease in mice.

There is a growing interest in the detection of subtle changes in cardiovascular physiology in response to viral infection to develop better disease surveillance strategies. This is not only important for earlier diagnosis and better prognosis of symptomatic carriers but also useful to diagnose asymptomatic carriers of the virus. Previous studies provide strong evidence of an association between inflammatory biomarker levels and both blood pressure (BP) and heart rate (HR) during infection. The identification of novel biomarkers during an inflammatory event could significantly improve predictions for cardiovascular events. Thus, we evaluated changes in cardiovascular physiology induced in A/Puerto Rico/8/34 (PR8) influenza infections in female and male C57BL/6J mice and compared them with the traditional method of influenza disease detection using body weight (BW). Using radiotelemetry, changes in BP, HR, and activity were studied. Change in BW of infected females was significantly decreased from 5 to 13 days postinfection (dpi), yet alterations in normal physiology including loss of diurnal rhythm and reduced activity was observed starting at about 3 dpi for HR and 4 dpi for activity and BP; continuing until about 13 dpi. In contrast, males had significantly decreased BW 8 to 12 dpi and demonstrated altered physiological measurements for a shorter period compared with females with a reduction starting at 5 dpi for activity, 6 dpi for BP, and 7 dpi for HR until about 12 dpi, 10 dpi, and 9 dpi, respectively. Finally, females and males exhibited different patterns of inflammatory maker expression in lungs at peak disease by analyzing bulk RNA-sequencing data for lungs and Bio-plex cytokine assay for blood collected from influenza-infected and naïve C57BL/6J female and male mice at 7 dpi. In total, this study provides insight into cardiovascular changes and molecular markers to distinguish sex differences in peak disease caused by influenza virus infection.NEW & NOTEWORTHY This study performed longitudinal cardiovascular measurements of influenza viral infection and identified sex difference in both physiological and molecular markers at peak disease.

Race and Sex Differences in Vital Signs Associated with COVID-19 and Flu Diagnoses in Mississippi.

Early detection of viral infections, such as COVID-19 and flu, have potential to reduce risk of morbidity, mortality, and disease transmission through earlier intervention strategies. For example, detecting changes in vital signs have the potential to more rapidly diagnose respiratory virus diseases. The objective of this study was to utilize the University of Mississippi Medical Center's extensive clinical database (EPIC) to investigate associations between temperature, pulse rate, blood pressure (BP), and respiration rate in COVID-19 and flu diagnosed patients. Data from 1,363 COVID-19 (March 3, 2020, to February 27, 2021) and 507 flu (October 1, 2017, to September 30, 2018) diagnosed patients with reported demographic dimensions (age, first race, and sex) and office visit dimensions (BMI, diastolic BP, pulse rate, respiration rate, systolic BP, and temperature) was obtained, including day of diagnosis and additional encounter visits 60 days before and after first unique diagnosis. Patients with COVID-19 or flu were disproportionately obese, with 93% of COVID-19 and 79% of flu patients with BMI ≥ 30. Most striking, Black women 50-64 years of age disproportionately carried the burden of disease. At the time of diagnosis, temperature was significantly increased for all patients, yet pulse rate was only significantly increased for flu diagnosis, and BP was not significantly different in either. Our findings show the need for more complete demographic and office visit dimension data from patients during epidemic and pandemic events and support further studies needed to understand association between vital signs and predicting respiratory disease.

Cellular co-infection can modulate the efficiency of influenza A virus production and shape the interferon response.

During viral infection, the numbers of virions infecting individual cells can vary significantly over time and space. The functional consequences of this variation in cellular multiplicity of infection (MOI) remain poorly understood. Here, we rigorously quantify the phenotypic consequences of cellular MOI during influenza A virus (IAV) infection over a single round of replication in terms of cell death rates, viral output kinetics, interferon and antiviral effector gene transcription, and superinfection potential. By statistically fitting mathematical models to our data, we precisely define specific functional forms that quantitatively describe the modulation of these phenotypes by MOI at the single cell level. To determine the generality of these functional forms, we compare two distinct cell lines (MDCK cells and A549 cells), both infected with the H1N1 strain A/Puerto Rico/8/1934 (PR8). We find that a model assuming that infected cell death rates are independent of cellular MOI best fits the experimental data in both cell lines. We further observe that a model in which the rate and efficiency of virus production increase with cellular co-infection best fits our observations in MDCK cells, but not in A549 cells. In A549 cells, we also find that induction of type III interferon, but not type I interferon, is highly dependent on cellular MOI, especially at early timepoints. This finding identifies a role for cellular co-infection in shaping the innate immune response to IAV infection. Finally, we show that higher cellular MOI is associated with more potent superinfection exclusion, thus limiting the total number of virions capable of infecting a cell. Overall, this study suggests that the extent of cellular co-infection by influenza viruses may be a critical determinant of both viral production kinetics and cellular infection outcomes in a host cell type-dependent manner.

A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.

Sequencing Framework for the Sensitive Detection and Precise Mapping of Defective Interfering Particle-Associated Deletions across Influenza A and B Viruses.

The mechanisms and consequences of defective interfering particle (DIP) formation during influenza virus infection remain poorly understood. The development of next-generation sequencing (NGS) technologies has made it possible to identify large numbers of DIP-associated sequences, providing a powerful tool to better understand their biological relevance. However, NGS approaches pose numerous technical challenges, including the precise identification and mapping of deletion junctions in the presence of frequent mutation and base-calling errors, and the potential for numerous experimental and computational artifacts. Here, we detail an Illumina-based sequencing framework and bioinformatics pipeline capable of generating highly accurate and reproducible profiles of DIP-associated junction sequences. We use a combination of simulated and experimental control data sets to optimize pipeline performance and demonstrate the absence of significant artifacts. Finally, we use this optimized pipeline to reveal how the patterns of DIP-associated junction formation differ between different strains and subtypes of influenza A and B viruses and to demonstrate how these data can provide insight into mechanisms of DIP formation. Overall, this work provides a detailed roadmap for high-resolution profiling and analysis of DIP-associated sequences within influenza virus populations.IMPORTANCE Influenza virus defective interfering particles (DIPs) that harbor internal deletions within their genomes occur naturally during infection in humans and during cell culture. They have been hypothesized to influence the pathogenicity of the virus; however, their specific function remains elusive. The accurate detection of DIP-associated deletion junctions is crucial for understanding DIP biology but is complicated by an array of technical issues that can bias or confound results. Here, we demonstrate a combined experimental and computational framework for detecting DIP-associated deletion junctions using next-generation sequencing (NGS). We detail how to validate pipeline performance and provide the bioinformatics pipeline for groups interested in using it. Using this optimized pipeline, we detect hundreds of distinct deletion junctions generated during infection with a diverse panel of influenza viruses and use these data to test a long-standing hypothesis concerning the molecular details of DIP formation.

Flu Shows the Power of Diversity.

Morphological variation is a common yet poorly understood feature of influenza virus populations. Vahey and Fletcher reveal that the production of physically and phenotypically heterogeneous particles is an inherent feature of the influenza assembly process that may promote virus survival in challenging environments.

Five life stage-specific transcriptome assemblies for the reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.

Feral Swine in the United States Have Been Exposed to both Avian and Swine Influenza A Viruses.

Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During the period from 2010 to 2013, 8,239 serum samples were collected from feral swine across 35 U.S. states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 tested virus, whereas the other 23 did not cross-react with any tested virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine had been exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs.IMPORTANCE There are more than 5 million feral swine distributed across at least 35 states in the United States. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock, and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine-adapted IAVs. Our findings suggest that feral swine have been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

Detection of Antigenic Variants of Subtype H3 Swine Influenza A Viruses from Clinical Samples.

A large population of genetically and antigenically diverse influenza A viruses (IAVs) are circulating among the swine population, playing an important role in influenza ecology. Swine IAVs not only cause outbreaks among swine but also can be transmitted to humans, causing sporadic infections and even pandemic outbreaks. Antigenic characterizations of swine IAVs are key to understanding the natural history of these viruses in swine and to selecting strains for effective vaccines. However, influenza outbreaks generally spread rapidly among swine, and the conventional methods for antigenic characterization require virus propagation, a time-consuming process that can significantly reduce the effectiveness of vaccination programs. We developed and validated a rapid, sensitive, and robust method, the polyclonal serum-based proximity ligation assay (polyPLA), to identify antigenic variants of subtype H3N2 swine IAVs. This method utilizes oligonucleotide-conjugated polyclonal antibodies and quantifies antibody-antigen binding affinities by quantitative reverse transcription-PCR (RT-PCR). Results showed the assay can rapidly detect H3N2 IAVs directly from nasal wash or nasal swab samples collected from laboratory-challenged animals or during influenza surveillance at county fairs. In addition, polyPLA can accurately separate the viruses at two contemporary swine IAV antigenic clusters (H3N2 swine IAV-α and H3N2 swine IAV-ß) with a sensitivity of 84.9% and a specificity of 100.0%. The polyPLA can be routinely used in surveillance programs to detect antigenic variants of influenza viruses and to select vaccine strains for use in controlling and preventing disease in swine.

Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans.

Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galß, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galß1-4GlcNAc substructures; Galß and GlcNAcß terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays.

Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples.