Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection

Background: Respiratory syncytial virus (RSV) is the major viral driver of a global pediatric respiratory disease burden disproportionately borne by the poor1. Thus, RSV, like SARS-CoV-2, combines with congenital and environmental and host-history-dependent factors to create a spectrum of disease with greatest severity most frequently occurring in those least able to procure treatment. Methods: Here we apply whole genome sequencing and a suite of other molecular biological techniques to survey host-virus dynamics in infections of two distinct cell lines (HEp2 and A549) with four strains representative of known RSV genetic diversity. Results: We observed non-gradient patterns of RSV gene expression and a single major difference in transcriptional readthrough correlating with a deep split in the RSV phylogenetic tree. We also observed increased viral replication in HEp2 cells along with a pro-inflammatory host-response; and decreased viral replication in A549 cells with a more potent antiviral response in host gene expression and levels of secreted cytokines. Conclusions: Our findings suggest HEp2 and A549 cell lines can be used as complementary models of host response leading to more or less severe RSV disease. In vitro perturbations inspired by actual environmental and host-history-dependent factors associated with greater disease can be tested for their ability to shift the antiviral response of A549 cells to the more pro-inflammatory response of HEp2 cells. Such studies would help illuminate the tragic costs of poverty and suggest public health-level interventions to reduce the global disease burden from RSV and other respiratory viruses

Wastewater pandemic preparedness: Toward an end-to-end pathogen monitoring program

Molecular analysis of public wastewater has great potential as a harbinger for community health and health threats. Long-used to monitor the presence of enteric viruses, in particular polio, recent successes of wastewater as a reliable lead indicator for trends in SARS-CoV-2 levels and hospital admissions has generated optimism and emerging evidence that similar science can be applied to other pathogens of pandemic potential (PPPs), especially respiratory viruses and their variants of concern (VOC). However, there are substantial challenges associated with implementation of this ideal, namely that multiple and distinct fields of inquiry must be bridged and coordinated. These include engineering, molecular sciences, temporal-geospatial analytics, epidemiology and medical, and governmental and public health messaging, all of which present their own caveats. Here, we outline a framework for an integrated, state-wide, end-to-end human pathogen monitoring program using wastewater to track viral PPPs

Microbiota of dominant Atlantic copepods: Pleuromamma sp. as a host to a betaproteobacterial symbiont

Copepods are the most abundant zooplankton group in the ocean and play a pivotal role as grazers of microorganisms and prey for larger animals. Furthermore, they are major contributors to the pool of dissolved organic material in the pelagic and therefore play an important role in the microbial loop. Although biology of copepods has been under investigation for more than a century, few studies have looked at the relationship between copepods and their associated bacteria. Could copepods be perceived as distinctive microbial hotspots in nutrient poor pelagic environment?The microbiota of three Pleuromamma species, an abundant genus of copepods that migrate vertically from surface waters to several hundred meters water depth and back on a daily basis was investigated using various molecular and morphological techniques. The focus was on the differences in the bacterial community composition of these copepods along the Atlantic Meridional Transect, which traverses major oceanic biomes such as the subtropical gyres and the equatorial convergence region. Additionally, the community structure and stable isotope composition of the likely microplankton food source as well as the Pleuromamma copepods was assessed along the same cruise transect.Sequencing of 16S rRNA tag libraries derived from individual Pleruromamma copepods showed a broad diversity of Bacteria associated with these copepods. These bacterial communities were uniform across the oceanic provinces and Pleuromamma species. These results were reflected in the uniformity of the prey community composition, however there was an indication that Pleuromamma copepods from different regions in the Atlantic rely on different food sources. Fluorescence in situ hybridisation and electron microscopy showed the presence of bacteria in the midgut region of the copepod guts and more specifically a high abundance of Betaproteobacteria.The bacterial community of a dominant copepod species in the Atlantic has previously not been studied on such a large spatial scale and sample size. This study shows that the same bacterial taxa were associated with Pleuromamma copepods inhabiting distinct oceanic regions. Moreover, a betaproteobacterial genus not present in the water-column appears to be closely associated with Pleuromamma

Evaluation of reference genes for RT-qPCR expression studies in hop (Humulus lupulus L.) during infection with vascular pathogen verticillium albo-atrum.

Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses

Primer sequences of 23 reference genes that were designed for qPCR amplification.

a<p>Unigene accession number according to the SOL Genomics Network (Solanaceae Genome Project - SOL) [<a href="http://www.sgn.cornell.edu/content/coffee.pl" target="_blank">http://www.sgn.cornell.edu/content/coffee.pl</a>].</p

Pairwise variation (V) between the normalization factors (NFn and NFn+1) to determine the optimal number of reference genes.

<p>V2/3 represents the pairwise variation between the NF values estimated for the two genes YLS8 and DRH1 and the NF values estimated for the three genes, YLS8, DRH1 and TIP41, and adding all 23 genes in the same way.</p

Expression data displayed as Ct values for each reference gene in all 12 samples.

<p>The figure summarizes data through 5 statistics: the markers in the box indicate average cycle threshold (Ct) values, squares indicate the presence of outliers, the box corresponding to an individual reference gene indicates the 25% and 75% percentiles, whiskers represent the maximum and minimum values and the median is depicted by the line across the box.</p