Limited Replication Research Articles

Heterogeneous Effects of Urban Land Titling: A Replication of ‘Property Rights for the Poor’

We replicate the analysis of Sebastian Galiani and Ernesto Shargrodsky’s (GS) influential 2010 article ‘Property rights for the poor: effects of land titling’. GS use a natural experiment in a poor urban area of Buenos Aires to find that land titling increases housing investment, reduces household size, and increases child education, but does not do so through increased use of credit. The original questionnaires and raw data are not available and the existing variables provided by GS allow only a limited replication analysis. Despite these limitations, we successfully reproduce the original findings published by GS, and find these results are robust to alternative specifications. We also find heterogeneous effects regarding gender and education level of the original squatter.

Transcriptional gene silencing limits CXCR4-associated depletion of bone marrow CD34+ cells in HIV-1 infection.

Hematological abnormalities that include changes in bone marrow, such as in anemia and pancytopenia, are common among HIV-infected patients, particularly in the advanced stage of disease. Such abnormalities may be caused by a reduced bone marrow function for hematopoiesis. The aim of this study was to determine whether transcriptional gene silencing can help to preserve the hosts' hematopoietic potential in addition to peripheral CD4+ T cells against CCR5-tropic HIV infection. NOD/SCID/JAK3null (NOJ) mice were transplanted with human cord-derived CD34+ cells with or without transduction with a lentiviral vector expressing a promoter-targeting shRNA called PromA. At 16 weeks after transplantation, mice engrafted with CD34+ cells were infected with CCR5-tropic HIV-1JRFL. At week 2 postinfection, HIV replication was observed in peripheral blood mononuclear cells and splenocytes. In mice transplanted with unmanipulated CD34+ cells, viral replication was accompanied by a loss of peripheral/spleen CD4+CCR5+ T cells. Interestingly, bone marrow CD34+ cells in HIV-infected mice were also depleted, but in a CXCR4-associated manner. Conversely, the lentiviral transfer of PromA in CD34+ cells prior to transplantation rendered the humanized NOJ mice resistant to HIV replication in CD4+ T cells, resulting in better preservation of peripheral/spleen CD4+CCR5+ T cells and bone marrow CD34+ cells at 2 weeks after infection. These results indicate that stable gene transfer of PromA to hematopoietic stem cells not only limited HIV replication but also led to preservation of different subsets of hematopoietic cells, including bone marrow stem/progenitor cells and CD4+ T cells.

Identifying drivers of spatial variation in occupancy with limited replication camera trap data

Occupancy models are widely used in camera trap studies to analyze species presence, abundance, and geographic distribution, among other important ecological quantities. These models account for imperfect detection using a latent variable to distinguish between true presence/absence and observed detection of a species. Under certain experimental setups, parameter estimation in a latent variable framework can be challenging. Several studies have issued guidelines on the number of independent replicated observations (surveys) needed for each unchanging occupancy field (season) to ensure reliable estimation. In this paper, we present a spatio-temporal occupancy model, and show through a simulation study that it can be fit to data obtained from a single survey per season, so long as the number of seasons is sufficiently large. We include an application using camera-trap data on the Thomson's gazelle in the Serengeti in Tanzania.

A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks.

Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses, such as Japanese encephalitis virus, West Nile virus, and Bagaza virus, is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks, while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks.IMPORTANCE Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis

Herpes simplex virus 2 (HSV-2) causes a persistent, lifelong infection that increases risk for sexually transmitted infection acquisition. Both the lack of a vaccine and the need for chronic suppressive therapies to control infection presents the need to further understand immune mechanisms in response to acute HSV-2 infection. The IL-36 cytokines are recently identified members of the IL-1 family and function as inflammatory mediators at epithelial sites. Here, we first used a well-characterized three-dimensional (3-D) human vaginal epithelial cell (VEC) model to understand the role of IL-36γ in the context of HSV-2 infection. In 3-D VEC, IL-36γ is induced by HSV-2 infection, and pretreatment with exogenous IL-36γ significantly reduced HSV-2 replication. To assess the impact of IL-36γ treatment on HSV-2 disease pathogenesis, we employed a lethal genital infection model. We showed that IL-36γ treatment in mice prior to lethal intravaginal challenge significantly limited vaginal viral replication, delayed disease onset, decreased disease severity, and significantly increased survival. We demonstrated that IL-36γ treatment transiently induced pro-inflammatory cytokines, chemokines, and antimicrobial peptides in murine lower female reproductive tract (FRT) tissue and vaginal lavages. Induction of the chemokines CCL20 and KC in IL-36γ treated mice also corresponded with increased polymorphonuclear (PMN) leukocyte infiltration observed in vaginal smears. Altogether, these studies demonstrate that IL-36γ drives the transient production of immune mediators and promotes PMN recruitment in the vaginal microenvironment that increases resistance to HSV-2 infection and disease. Our data indicate that IL-36γ may participate as a key player in host defense mechanisms against invading pathogens in the FRT.

Immunization by Replication-Competent Controlled Herpesvirus Vectors.

Replication-competent controlled virus vectors were derived from the virulent herpes simplex virus 1 (HSV-1) wild-type strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is coactivated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wild-type virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors-which may be regarded as equivalents of inactivated vaccines-induced detectable protection against lethality caused by wild-type virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and a second immunization resulted in complete protection. Once activated, vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus, as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a severalfold enhancement of these immune responses.IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, the necessary safety should be provided by placing replication of the pathogen under stringent control and by activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above-mentioned hypothesis and suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.

Human Genetics of Obesity and Type 2 Diabetes Mellitus: Past, Present, and Future.

Type 2 diabetes mellitus (T2D) and obesity already represent 2 of the most prominent risk factors for cardiovascular disease, and are destined to increase in importance given the global changes in lifestyle. Ten years have passed since the first round of genome-wide association studies for T2D and obesity. During this decade, we have witnessed remarkable developments in human genetics. We have graduated from the despair of candidate gene-based studies that generated few consistently replicated genotype-phenotype associations, to the excitement of an exponential harvest of loci robustly associated with medical outcomes through ever larger genome-wide association study meta-analyses. As well as discovering hundreds of loci, genome-wide association studies have provided transformative insights into the genetic architecture of T2D and other complex traits, highlighting the extent of polygenicity and the tiny effect sizes of many common risk alleles. Genome-wide association studies have also provided a critical starting point for discovering new biology relevant to these traits. Expectations are high that these discoveries will foster development of more effective strategies for intervention, through optimization of precision medicine approaches. In this article, we review current knowledge and provide suggestions for the next steps in genetic research for T2D and obesity. We focus on four areas relevant to precision medicine: genetic architecture, pharmacogenetics and other gene-environment interactions, mechanistic inference, and drug development. As we describe, the genetic architecture of complex traits has major implications for the prospects of precision medicine, rendering some anticipated approaches decidedly unrealistic. We highlight obstacles to the translation of human genetic findings into mechanism inference but are optimistic that, as these are overcome, there is untapped potential for novel drugs and more effective strategies for treating and preventing T2D and obesity.

A Systematic Review of Commercial Cognitive Training Devices: Implications for Use in Sport

Background: Cognitive training (CT) aims to develop a range of skills, like attention and decision-making, through targeted training of core cognitive functions. While CT can target context specific skills, like movement anticipation, much CT is domain general, focusing on core abilities (e.g., selective attention) for transfer to a range of real-world tasks, such as spotting opponents. Commercial CT (CCT) devices are highly appealing for athletes and coaches due to their ease of use and eye-catching marketing claims. The extent to which this training transfers to performance in the sporting arena is, however, unclear. Therefore, this paper sought to provide a systematic review of evidence for beneficial training effects of CCT devices and evaluate their application to sport.Methods: An extensive search of electronic databases (PubMed, PsychInfo, GoogleScholar, and SportDiscus) was conducted to identify peer-reviewed evidence of training interventions with commercially available CT devices. Forty-three studies met the inclusion criteria and were retained for quality assessment and synthesis of results. Seventeen studies assessed transfer effects beyond laboratory cognitive tests, but only 1 directly assessed transfer to a sporting task.Results: The review of evidence showed limited support for far transfer benefits from CCT devices to sporting tasks, mainly because studies did not target the sporting environment. Additionally, a number of methodological issues with the CCT literature were identified, including small sample sizes, lack of retention tests, and limited replication of findings by researchers independent of the commercial product. Therefore, evidence for sporting benefits is currently limited by the paucity of representative transfer tests and a focus on populations with health conditions.Conclusions: Currently there is little direct evidence that the use of CCT devices can transfer to benefits for sporting performance. This conclusion, however, stems more from a lack of experimental studies in the sporting field and a lack of experimental rigor, rather than convincing null effects. Subsequently, there is an opportunity for researchers to develop more reliable findings in this area through systematic assessment in athletic populations and major methodological improvements.

A Decade of Blood Biomarkers for Alzheimer's Disease Research: An Evolving Field, Improving Study Designs, and the Challenge of Replication.

Blood-based biomarkers represent a less invasive and potentially cheaper approach for aiding Alzheimer’s disease (AD) detection compared with cerebrospinal fluid and some neuroimaging biomarkers. Acknowledging that many in the field have made great progress, here we review some of the work that our group has pursued to identify and validate blood-based proteomic biomarkers through both case control and AD pathology endophenotype-based approaches. Our focus is primarily to identify a minimally invasive and hopefully cost-effective blood-based biomarker to reduce screen failure in clinical trials where participants have prodromal or even pre-clinical disease. We summarize some of the key findings and approaches taken in these biomarker studies, while addressing the main challenges, including that of limited replication in the field, and discuss opportunities for biomarker development.

Pharmacogenetic Effects of Naltrexone in Individuals of East Asian Descent: Human Laboratory Findings from a Randomized Trial.

Genetic variation in the endogenous opioid system has been identified as 1 potential source of individual variability in naltrexone treatment outcomes. The majority of naltrexone pharmacogenetic studies have focused on a particular single nucleotide polymorphism (SNP) of the mu-opioid receptor gene (OPRM1; rs1799971; commonly known as the Asn40Asp SNP) in Caucasian samples with decidedly mixed results. The goal of this study was to test the pharmacogenetic effects of naltrexone on subjective response to alcohol and self-administration of alcohol in individuals of East Asian descent. We hypothesized that naltrexone, compared with placebo, would potentiate the aversive and sedative effects of alcohol and reduce alcohol self-administration to a greater extent in Asp40 carriers. Participants (N=77; Asn40Asn, n=29; Asn40Asp, n=34, and Asp40Asp, n=14) completed 2 double-blinded and counterbalanced experimental sessions: one after taking naltrexone (50mg/d) for 5days and one after taking matched placebo for 5days. In each experimental session, participants received a priming dose of intravenous alcohol up to the breath alcohol concentration target of 0.06g/dl which was immediately followed by an alcohol self-administration period (1hour). There were no pharmacogenetic effects observed for alcohol-induced stimulation, sedation, craving for alcohol, or alcohol self-administration in the laboratory. During the self-administration period, Asp40 carriers consumed fewer drinks and had a longer latency to first drink as compared to Asn40 homozygotes. These findings in East Asians add to the mixed literature on naltrexone pharmacogenetics from predominantly Caucasian samples and highlight the complexity of these effects and their overall limited replicability. It is plausible that a consistent pharmacogenetic effect in tightly controlled preclinical and experimental medicine models "fades" in more complex and heterogeneous settings and samples.

Statistical analysis of multi-dimensional, temporal gene expression of stem cells to elucidate colony size-dependent neural differentiation.

High throughput gene expression analysis using qPCR is commonly used to identify molecular markers of complex cellular processes. However, statistical analysis of multi-dimensional, temporal gene expression data is complicated by limited biological replicates and large number of measurements. Moreover, many available statistical tools for analysis of time series data assume that the data sequence is static and does not evolve over time. With this assumption, the parameters used to model the time series are fixed and thus, can be estimated by pooling data together. However, in many cases, dynamic processes of biological systems involve abrupt changes at unknown time points, making the assumption of stationary time series break down. We addressed this problem using a combination of statistical methods including hierarchical clustering, change point detection, and multiple testing. We applied this multi-step method to multi-dimensional, temporal gene expression data that resulted from our study of colony size-dependent neural cell differentiation of stem cells. The gene expression data were time series as the observations were recorded sequentially over time. Hierarchical clustering segregated the genes into three distinct clusters based on their temporal expression profiles; change point detection identified specific time points at which the entire dataset was divided into several homogenous subsets to allow a separate analysis of each subset; and multiple testing procedure identified the differentially expressed genes in each cluster within each subset of data. We established that our multi-step approach pinpoints specific sets of genes that underlie colony size-mediated neural differentiation of stem cells and demonstrated its advantages over conventional parametric and non-parametric tests that do not take into account temporal dynamics of the data. Importantly, our proposed approach is broadly applicable to any multivariate data sets of limited sample size from high throughput and high content screening such as in drug and biomarker discovery studies.

Robust determination of differential abundance in shotgun proteomics using nonparametric statistics.

Label-free shotgun mass spectrometry enables the detection of significant changes in protein abundance between different conditions. Due to often limited cohort sizes or replication, large ratios of potential protein markers to number of samples, as well as multiple null measurements pose important technical challenges to conventional parametric models. From a statistical perspective, a scenario similar to that of unlabeled proteomics is encountered in genomics when looking for differentially expressed genes. Still, the difficulty of detecting a large fraction of the true positives without a high false discovery rate is arguably greater in proteomics due to even smaller sample sizes and peptide-to-peptide variability in detectability. These constraints argue for nonparametric (or distribution-free) tests on normalized peptide values, thus minimizing the number of free parameters, as well as for measuring significance with permutation testing. We propose such a procedure with a class-based statistic, no parametric assumptions, and no parameters to select other than a nominal false discovery rate. Our method was tested on a new dataset which is available via ProteomeXchange with identifier PXD006447. The dataset was prepared using a standard proteolytic digest of a human protein mixture at 1.5-fold to 3-fold protein concentration changes and diluted into a constant background of yeast proteins. We demonstrate its superiority relative to other approaches in terms of the realized sensitivity and realized false discovery rates determined by ground truth, and recommend it for detecting differentially abundant proteins from MS data.

Neurocognitive Impairment and Associated Genetic Aspects in HIV Infection.

HIV enters the central nervous system (CNS) early after infection. HIV-associated neurocognitive disorders (HAND) remain a serious complication of HIV infection despite available antiretroviral therapy (ART). Neurocognitive deficits observed in HAND are heterogeneous, suggesting a variability in individuals' susceptibility or resiliency to the detrimental CNS effects of HIV infection. This chapter reviews primary host genomic changes (immune-related genes, genes implicated in cognitive changes in primary neurodegenerative diseases), epigenetic mechanisms, and genetic interactions with ART implicated in HIV progression or HAND/neurocognitive complications of HIV. Limitations of the current findings include diversity of the HAND phenotype and limited replication of findings across cohorts. Strategies to improve the precision of future (epi)genetic studies of neurocognitive consequences of HIV infection are offered.

Alternative Values-Based 'Recipes' for Life Satisfaction: German Results with an Australian Replication

In most research on Life Satisfaction (LS), it is assumed that the covariates of high and low LS are the same for everyone, or at least everyone in the West. In this paper, analysing data from the German Socio-Economic Panel, with a limited replication based on Australian panel data, we estimate models of alternative ‘recipes’ for LS. There appear to be at least four distinct ‘recipes’, which are primarily based on the values of different population sub-sets. These values are: altruistic values, family values, materialistic values and religious values. By a ‘recipe’ for LS we mean a linked set of values, behavioural choices and domain satisfactions, which appear to be held together by a person’s values, and which prove to have substantial effects on LS. Our German and Australian evidence indicates that individuals who follow recipes based on altruistic, family or religious values record above average long term LS, whereas the materialistic values ‘recipe’ is associated with below average LS.

Comparison of five methods for parameter estimation under Taylor’s power law

Taylor’s power law (TPL) can be applied to the mean-variance relationship for various quantities, e.g., population densities over space and time, biomass of plants in the growth processes, developmental rates and growth rates of arthropods at different temperatures, etc. When TPL holds in the grouped data (e.g., the biomass of plants at different investigation times with limited replicates at each investigation), we must consider the heterocedasticity when carrying out the parameter estimation for a given mathematical model. We propose two new target functions based on TPL and attempt to more accurately estimate the model parameter(s). By capturing the mean-variance relationship, the accuracy of parameter estimation and the power of hypothesis testing are improved compared to the traditional methods which assume homogeneous variance. Five approaches, including ordinary least squares (OLS), chi-squared (χ2), weighted least squares (WLS), psi-squared (ψ2) and maximum likelihood estimation (MLE), are compared under both linear and nonlinear scenarios regarding the prediction accuracy using both computer simulations and experimental data. We further simulate irregular measurements on the predictors to examine the robustness of parameter estimation. In computer simulations, psi-squared, WLS, and MLE outperform OLS and chi-squared with respect to parameter estimation. In the simulation study of irregular measurements, psi-squared and MLE outperform WLS when the sample mean is smaller than the population mean. On the other hand, WLS outperforms psi-squared and MLE when the sample mean is larger than the population mean. Psi-squared has an advantage over MLE when large irregular deviations are present. This study strongly suggests using psi-squared and WLS in place of OLS or chi-squared for both linear and nonlinear regressions, the choice of method depending on the observations.

IRF9 Prevents CD8+ T Cell Exhaustion in an Extrinsic Manner during Acute Lymphocytic Choriomeningitis Virus Infection.

Effective CD8+ T cell responses play an important role in determining the course of a viral infection. Overwhelming antigen exposure can result in suboptimal CD8+ T cell responses, leading to chronic infection. This altered CD8+ T cell differentiation state, termed exhaustion, is characterized by reduced effector function, upregulation of inhibitory receptors, and altered expression of transcription factors. Prevention of overwhelming antigen exposure to limit CD8+ T cell exhaustion is of significant interest for the control of chronic infection. The transcription factor interferon regulatory factor 9 (IRF9) is a component of type I interferon (IFN-I) signaling downstream of the IFN-I receptor (IFNAR). Using acute infection of mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong, we show here that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and IFN-I and by controlling levels of IRF7, a transcription factor essential for IFN-I production. Infection of IRF9- or IFNAR-deficient mice led to a loss of early restriction of viral replication and impaired antiviral responses in dendritic cells, resulting in CD8+ T cell exhaustion and chronic infection. Differences in the antiviral activities of IRF9- and IFNAR-deficient mice and dendritic cells provided further evidence of IRF9-independent IFN-I signaling. Thus, our findings illustrate a CD8+ T cell-extrinsic function for IRF9, as a signaling factor downstream of IFNAR, in preventing overwhelming antigen exposure resulting in CD8+ T cell exhaustion and, ultimately, chronic infection.IMPORTANCE During early viral infection, overwhelming antigen exposure can cause functional exhaustion of CD8+ T cells and lead to chronic infection. Here we show that the transcription factor interferon regulatory factor 9 (IRF9) plays a decisive role in preventing CD8+ T cell exhaustion. Using acute infection of mice with LCMV strain Armstrong, we found that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and Irf7, encoding a transcription factor crucial for type I interferon (IFN-I) production, as well as by controlling the levels of IFN-I. Infection of IRF9-deficient mice led to a chronic infection that was accompanied by CD8+ T cell exhaustion due to defects extrinsic to T cells. Our findings illustrate an essential role for IRF9, as a mediator downstream of IFNAR, in preventing overwhelming antigen exposure causing CD8+ T cell exhaustion and leading to chronic viral infection.

Productive replication of avian influenza viruses in chicken endothelial cells is determined by hemagglutinin cleavability and is related to innate immune escape

Endotheliotropism is a hallmark of gallinaceous poultry infections with highly pathogenic avian influenza (HPAI) viruses and a feature that distinguishes HPAI from low pathogenic avian influenza (LPAI) viruses. Here, we used chicken aortic endothelial cells (chAEC) as a novel in vitro infection model to assess the susceptibility, permissiveness, and host response of chicken endothelial cells (EC) to infections with avian influenza (AI) viruses. Our data show that productive replication of AI viruses in chAEC is critically determined by hemagglutinin cleavability, and is thus an exclusive trait of HPAI viruses. However, we provide evidence for a link between limited (i.e. trypsin-dependent) replication of certain LPAI viruses, and the viruses’ ability to dampen the antiviral innate immune response in infected chAEC. Strikingly, this cell response pattern was also detected in HPAI virus-infected chAEC, suggesting that viral innate immune escape might be a prerequisite for robust AI virus replication in chicken EC.

Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.

Human intestinal organoids express histo-blood group antigens, bind norovirus VLPs, and support limited norovirus replication

Through pluripotent stem cell (PSC) technology, human intestinal organoids (HIOs) with remarkably similarity to the fetal intestine in cellular composition, architecture, and absorptive/secretory functions have been successfully developed, providing a useful in vitro model system to study the structure and function of human congenital gut and intestinally related diseases. We report here the usefulness of HIOs as a model system to study intestinal carbohydrate expression, virus-host interaction, and replication of human noroviruses (huNoVs). We found that fully developed HIOs express effectively various types 1 and 2 HBGAs, including Lewis, secretor, and nonsecretor antigens, distributing on the glycocalyx. Selected huNoV-like particles (VLPs) bound the glycocalyx of HIOs with matched HBGA phenotypes. Using GII.4 huNoV positive stool filtrates, we demonstrated limited huNoV replication in HIOs with corresponding HBGAs through detection of viral RNAs by RT-PCR and capsid antigens by immunostaining methods. Our data suggested that, after further improvements, HIOs can be a useful model to study intestinal glycan expression, huNoV-intestine interaction, and huNoV infection in the intestine.

Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans.

Bats are reservoir hosts of many important viruses that cause substantial disease in humans, including coronaviruses, filoviruses, lyssaviruses, and henipaviruses. Other than the lyssaviruses, they do not appear to cause disease in the reservoir bats, thus an explanation for the dichotomous outcomes of infections of humans and bat reservoirs remains to be determined. Bats appear to have a few unusual features that may account for these differences, including evidence of constitutive interferon (IFN) activation and greater combinatorial diversity in immunoglobulin genes that do not undergo substantial affinity maturation. We propose these features may, in part, account for why bats can host these viruses without disease and how they may contribute to the highly pathogenic nature of bat-borne viruses after spillover into humans. Because of the constitutive IFN activity, bat-borne viruses may be shed at low levels from bat cells. With large naive antibody repertoires, bats may control the limited virus replication without the need for rapid affinity maturation, and this may explain why bats typically have low antibody titers to viruses. However, because bat viruses have evolved in high IFN environments, they have enhanced countermeasures against the IFN response. Thus, upon infection of human cells, where the IFN response is not constitutive, the viruses overwhelm the IFN response, leading to abundant virus replication and pathology.