Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vectorborne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for widespread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

Continuous-time random walks (CTRWs) on discrete state spaces, ranging from regular lattices to complex networks, are ubiquitous across physics, chemistry, and biology. Models with coarse-grained states (for example, those employed in studies of molecular kinetics) or spatial disorder can give rise to memory and non-exponential distributions of waiting times and first-passage statistics. However, existing methods for analyzing CTRWs on complex energy landscapes do not address these effects. Here we use statistical mechanics of the nonequilibrium path ensemble to characterize first-passage CTRWs on networks with arbitrary connectivity, energy landscape, and waiting time distributions. Our approach can be applied to calculating higher moments (beyond the mean) of path length, time, and action, as well as statistics of any conservative or non-conservative force along a path. For homogeneous networks, we derive exact relations between length and time moments, quantifying the validity of approximating a continuous-time process with its discrete-time projection. For more general models, we obtain recursion relations, reminiscent of transfer matrix and exact enumeration techniques, to efficiently calculate path statistics numerically. We have implemented our algorithm in PathMAN (Path Matrix Algorithm for Networks), a Python script that users can apply to their model of choice. We demonstrate the algorithm on a few representative examples which underscore the importance of non-exponential distributions, memory, and coarse-graining in CTRWs.

We propose a novel Bayesian methodology which uses random walks for rapid inference of statistical properties of undirected networks with weighted or unweighted edges. Our formalism yields high-accuracy estimates of the probability distribution of any network node-based property, and of the network size, after only a small fraction of network nodes has been explored. The Bayesian nature of our approach provides rigorous estimates of all parameter uncertainties. We demonstrate our framework on several standard examples, including random, scale-free, and small-world networks, and apply it to study epidemic spreading on a scale-free network. We also infer properties of the large-scale network formed by hyperlinks between Wikipedia pages.

29Emerging and neglected diseases pose challenges as their biology is frequently poorly understood, and 30 genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing 31 technologies offer a promising approach to understand the molecular processes underlying these diseases. 32Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium and the 33 causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of 34 repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, 35 comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture 36 of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-37 transcriptional antisense regulation. We compared the host response to two clinical isolates and identified 38 distinct immune response networks that are up-regulated in response to each strain, leading to predictions 39 of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide 40 insight into the biology and host-pathogen interactions of a poorly characterized and genetically 41 intractable organism such as Ot. 42 43 Keywords: neglected and emerging pathogens, intracellular bacteria, dual RNA-seq, transcriptomics, 44 host-pathogen cell biology, bacterial virulence, antisense transcription. 45 4 Main Text 46 48 serious pathogens, whilst human population growth, globalization and increased travel have contributed 49 to the emergence of new pathogens and changing patterns of infectious disease. The biology of neglected 50 and emerging pathogens is often poorly understood, but is essential to developing therapeutic and 51 preventative strategies. Obligate intracellular pathogens present additional challenges, as many cause 52 diseases that are difficult to diagnose and are difficult to manipulate experimentally. 53 54 Obligate intracellular bacteria include the Rickettsiales, an order which includes the arthropod and 55 nematode symbiont Wolbachia as well as a number of human and veterinary pathogens. Orientia 56 tsutsugamushi (Ot, Class Alphaproteobacteria, Order Rickettsiales, Family Rickettsiaceae) causes the 57 mite-borne human disease scrub typhus, a leading cause of severe febrile illness in the Asia Pacific region 1 , 58 home to roughly two thirds of the world's population. Locally acquired cases in the Middle East and Latin 59America suggest that this disease may be more widespread than previously appreciated 2,3 . Under-60 recognition and under-reporting are a major problem in scrub typhus because unambiguous diagnosis is 61 difficult, and awareness is low amongst many clinicians. Symptoms are non-specific and include 62 headache, fever, rash, and lymphadenopathy beginning 7-14 days after inoculation via a feeding larval 63 stage mite. If untreated, this can progress to cause multiple organ failure and death. In the mite vector Ot 64 colonizes...

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