DOI: 10.5061/dryad.866t1g1zs
title: Orsay virus infection increases Caenorhabditis elegans resistance to heat-shock

description: The heat shock response plays a role in the immune defense against viruses
 across various organisms. Studies on model organisms show that inducing
 this response prior to viral exposure enhances host resistance to
 infections, while deficient responses make individuals more vulnerable.
 Moreover, viruses rely on components of the heat shock response for their
 own stability and viral infections improve thermal tolerance in plants,
 giving infected individuals an advantage in extreme conditions, which aids
 the virus in replication and transmission. Here, we examine the
 interaction between the nematode Caenorhabditis elegans and its natural
 pathogen the Orsay virus (OrV) under heat stress. We found that OrV
 infection leads to differential expression of heat-stress related genes,
 and infected populations showed increased resistance to heat shock. This
 resistance correlated with increased expression of argonautes alg-1 and
 alg-2, which are crucial for survival after heat shock and for OrV
 replication. Overall, our study suggests an environmental-dependent
 mutualistic relationship between the nematode and OrV, potentially
 expanding the animal’s ecological niche and providing the virus with extra
 opportunities for replication and adaptation to extreme conditions.
description: <strong>(a) Overlap of heat-stress related GO terms and OrV
 DEGs</strong> The differentially expressed genes
 upon OrV infection in wild-type animals were obtained from. The genes
 belonging to the GO term categories (<em>i</em>) cellular
 response to unfolded proteins, (<em>ii</em>) heat-shock
 binding proteins and (<em>iii</em>) response to heat were
 extracted from WormBase Ontology Browser. Only genes annotated as directly
 involved in each category were used. Genes that were differentially
 expressed upon OrV infection and belonged to any of these three GO term
 categories were selected. <strong>(b) Strain
 maintenance</strong> Nematodes were maintained at
 20 ºC on Nematode Growth Medium (NGM) plates seeded with
 <em>Escherichia coli </em>OP50 under standard conditions.
 ERT54 (<em>jyIs8 [pals-5p::GFP; myo-2p::mCherry]X</em> in an
 N2 Bristol background) served as wild-type. JU2624,
 (<em>mjIs228[myo-2p::mCherry::unc54; lys-3p::eGFP::tbb-2]
 </em>in a JU1580 background) was the natural isolate from which OrV
 was first identified. Both strains were generously provided by Prof. M.A.
 Félix. <strong>(c) OrV stock preparation and
 quantification</strong> JU2624 animals were
 inoculated with OrV isolate JUv1580 (a gift from Prof. M.A. Félix),
 allowed to grow for 5 days and then resuspended in M9 buffer (0.22 M
 KH<sub>2</sub>PO<sub>4</sub>, 0.42 M
 Na<sub>2</sub>HPO<sub>4</sub>, 0.85 M NaCl, 0.001
 M MgSO<sub>4</sub>), let stand for 15 min at room temperature,
 vortexed, and centrifuged for 2 min at 400 g. The supernatant was
 centrifuged twice at 21,000 g for 5 min and then passed through a 0.2 mm
 filter. RNA of the resulting viral stock was extracted using the Viral RNA
 Isolation kit (NZYTech). The concentration of viral RNA was determined by
 RT-qPCR and normalized using a standard curve. Primers used can be found
 in table 1. For the standard curve cDNA of OrV was
 obtained using Accuscript High Fidelity Reverse Transcriptase (Agilent)
 and reverse primers at the 3’ end of the virus (see table 1 for primers).
 Approximately 1000 bp of the 3’ end of RNA1 and RNA2 were amplified using
 forward primers containing 20 bp coding the T7 promoter and DreamTaq DNA
 Polymerase (ThermoFisher). The PCR products were gel-purified using MSB
 Spin PCRapace (Invitek Molecular) and an <em>in vitro</em>
 transcription was performed using T7 Polymerase (Merck). The remaining DNA
 was then degraded using DNAse I (Life Technologies). RNA concentration was
 determined by NanoDrop (ThermoFisher) and the number of molecules per µL
 was determined using the online tool EndMemo RNA Copy Number Calculator
 (https://endmemo.com/bio/dnacopynum.php).
 <strong>(d) Nematode synchronization and OrV
 inoculation</strong> In order to obtain
 synchronized populations of wild-type and JU2624 animals, plates with eggs
 were carefully washed with M9 buffer to remove larvae and adults but
 leaving the eggs behind. Plates were washed again using M9 buffer after 1
 h to collect larvae hatched within that time span. Synchronized nematode
 populations were inoculated with 4.92´10<sup>8</sup> copies of
 OrV –a concentration that leads to activation of the
 <em>pals-5p::GFP </em>reporter in ~50% of the animals– by
 pipetting the viral stock on top of the bacterial lawn containing the
 animals. Twenty-four hours post-inoculation (hpi)
 <em>pals-5p::GFP</em> negative animals were manually removed
 from infected plates and negative control plates were checked to confirm
 absence of <em>pals-5p::GFP </em>activation.
 <strong>(e) Heat-shock</strong>
 Synchronized population were shifted from a 20 ºC incubator to a
 37 ºC water bath at 48 hpi for 2 h. This heat-shock treatment results in
 ~50% mortality of control animals, and hereafter we will refer to it as
 semi-lethal. Plates were sealed with parafilm and placed bottom down in
 order to ensure a quick temperature shift. After the heat shock they were
 returned to 20 ºC (figure 1b). <strong>(f)
 Mortality assay</strong> Twenty-four hours
 post-heat-shock (hphs) mortality within the heat-shocked population was
 quantified. Animals were considered dead when they did not react to touch
 and had stopped pharyngeal pumping. Mortality was
 evaluated in four and eight independent full blocks for JU2624 and
 wild-type populations, respectively. The number of plaques (biological
 replicates) within each experimental condition within blocks varied
 between 2 and 13 (median 3).  Mean and standard deviation of mortality
 were estimated for each infection condition, nematode strain and
 experimental block. Data were analyzed using a meta-analysis approach for
 continuous data, with infection status considered as a fixed factor, using
 Cohen’s <em>d</em> (±1 standard error) as a normalized
 estimation of the effect size, and restricted maximum-likelihood for
 parameter’s estimation. These analyses were done using SPSS version
 29.0.1.1 (IBM Corp). <strong>(g) Quantification
 of <em>alg-1 </em>and <em>alg-2
 </em>expression</strong> To quantify
 expression levels of <em>alg-1 </em>and
 <em>alg-2,</em> wild-type control and infected animals were
 used. Nematodes were heat-shocked as described above and samples were
 taken before heat-shock, right after the heat-shock, and 2 and 8 hphs.
 Animals were collected with PBS 0.05% Tween and washed 3 times before
 freezing in liquid N<sub>2</sub>. RNA was extracted using
 Trizol as previously described. RT-qPCRs were performed using Power SYBR
 Green PCR Master Mix (Applied Biosystems) on an ABI StepOne Plus Real-time
 PCR System (Applied Biosystems). Ten ng of total RNA were loaded and
 samples were normalized to expression of <em>cdc-42</em>.
 Primers used can be found in table 1. Relative expression levels were
 computed using the DD<em>C<sub>T</sub></em>
 method. Fold-change values for each mutant genotype
 were independently fitted to a generalized linear model with a Normal
 distribution and identity function with infection status and hpsh
 incorporated as orthogonal random factors. The interaction between the two
 factors is reported in the text as an evaluation of the differences in
 temporal expression dynamics. These analyses were also done with SPSS
 version 29.0.1.1.
description: # Orsay virus infection increases *Caenorhabditis elegans* resistance to
 heat-shock
 [https://doi.org/10.5061/dryad.866t1g1zs](https://doi.org/10.5061/dryad.866t1g1zs) Excel files containing survival of infected and noninfected animals in control and heat-shock conditions.  Each file corresponds to a different experimental block.  Data necessary to reproduce Figure 1c. Excel file containing the expression of *alg-1* and *alg-2* argonaute genes. Data necessary to reproduce Figure 1d. ## Description of the data and file structure Excel file 1: Heat shock 210902.xlsx *C. elegans* strains JU2624 and ERT75 in control (+ M9 salts) and inoculated (+ OrV) treatments. Number of alive and dead animals. Probability of survival. Excel file 2: Heat shock 210820.xlsx *C. elegans* strains JU2624 and ERT75 in control (+ M9 salts) and inoculated (+ OrV) treatments. Number of alive and dead animals. Probability of survival. Excel file 3: Heat shock 210826.xlsx *C. elegans* strains JU2624 and ERT75 in control (+ M9 salts) and inoculated (+ OrV) treatments. Number of alive and dead animals. Probability of survival. Excel file 4: Heat shock 210827.xlsx *C. elegans* strains JU2624 and ERT75 in control (+ M9 salts) and inoculated (+ OrV) treatments. Number of alive and dead animals. Probability of survival. Excel file 5: Meta-analysis.xlsx Different worm genotypes, *P* value of the corresponding t-test comparing infected and non-infected heat-treated animals, log*P*-value, number of tests, weight, inverse weighted *P*-value. Raw 9, column C: Fisher's X^2 test for combining *P*-values from independent tests. Raw 10, column C: corresponding *P* value for the X^2 test. Raw 10, column F: harmonic mean *P* value. Excel file 6: Analysis *alg1* and2.xlsx Sheet 1: Analyzed data. Ct, Delta-Ct, average Delta-Ct, Delta-Delta-Ct, and expression level for endogenous control CDC-42 and ALG-1 and ALG-2 genes. Sheet 2: Raw Ct and Delta-Delta-Ct data n/a stands for not available data ## Sharing/Access information Links to other publicly accessible locations of the data: * [https://git.csic.es/SFElenaLab/heat-shock-and-orv-infection.](https://git.csic.es/SFElenaLab/heat-shock-and-orv-infection.) GO and DEGs used to prepare Figure 1a are from: * [https://doi.org/10.1101/2023.10.31.564947](https://doi.org/10.1101/2023.10.31.564947)