We would like to thank the UTMB imaging core facilities and Maxim Ivannikov for microscope assistance.
This work was supported by Public Health Service grant RO1AI093445 and RO1AI129198 awarded to SP, UTMB Commitment Fund P84373 to CH, and T32 AI007526 to EM.

1. Preparation of A549 cells and JUNV infection
<ol>
	<li>Seed 2 x 105 human lung epithelial A549 cells onto poly-D-lysine (PDL) coated glass coverslips in 12-well plates at 24 hours prior to infection.</li>
	<li>Prepare aliquots of 150 &#956;L of JUNV13 at a multiplication of infection (MOI) of 1.0 plaque forming unit per cell diluted in Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) media supplemented with 2% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S).</li>
	<li>Remove media from cell culture. Add virus inoculum on each well containing coverslip and incubate for 1.5 h at 37 &#176;C. Shake the plates every 15 min.</li>
	<li>Remove the virus inoculum, add 1 mL of DMEM supplemented with 5% FBS and 1% P/S. Incubate the plate at 37 &#176;C for desired time point.</li>
</ol>
2. Fixation and immunostaining
<ol>
	<li>Aspirate the media. Rinse the cells by adding 1 mL of phosphate buffered saline (PBS) supplemented with calcium and magnesium to each well.</li>
	<li>Remove PBS. Add 1 mL of methanol (MeOH) pre-chilled at -20 &#176;C and incubate at -20 &#176;C or on dry ice for 15 min.</li>
	<li>Remove MeOH.</li>
	<li>Add 1 mL of PBS to each well, and wash samples at 4 &#176;C with gentle rocking for 5 min. Repeat the wash for total of 4 times.</li>
	<li>Wash the fixed cells on coverslips in 1 mL of 0.2% t-octylphenoxypolyethoxyethanol for 5 min at 4 &#176;C, with gentle rocking.</li>
	<li>Add 1 mL of PBS to each well. Wash samples at 4 &#176;C for 5 min with gentle rocking. Repeat the washing step for total of 4 times.</li>
	<li>To detect dsRNA and MDA5, incubate samples in 200 &#956;L of primary antibodies diluted in 3% bovine serum albumin (BSA). Dilute the anti-dsRNA 9D5 antibody at a 1:2 dilution and dilute the anti-MDA-5 antibody at 1:250. Incubate with gentle rocking at 4 &#176;C overnight.</li>
	<li>Remove primary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.</li>
	<li>Add 200 &#956;L of secondary antibodies (1:2,000 dilution) diluted in 3% BSA and incubate at RT for 1 h.</li>
	<li>Remove secondary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.</li>
	<li>To detect JUNV nucleoprotein (NP) and RIG-I, add 200 &#956;L of conjugated antibodies diluted in 3% BSA. Dilute the conjugated anti-JUNV NP (AG-12) at 1:1,000 and incubate samples for 2 h at RT with gentle rocking. Dilute the conjugated anti-RIG-I antibody at 1:500 and incubate at 4 &#176;C overnight with gentle rocking.</li>
	<li>Remove conjugated antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.</li>
	<li>Counterstain the coverslips with DAPI (1:1,000) for 3 min with gentle rocking at RT.</li>
	<li>Wash the coverslips 3 times, each time for 5 min in 1 mL of 0.5% t-octylphenoxypolyethoxyethanol with gentle rocking at RT.</li>
	<li>Wash twice in 1 mL of PBS for 5 min each time with gentle rocking at RT.</li>
	<li>Wash once in 1 mL of ddH2O for 1 min at RT, rocking gently.</li>
	<li>Mount coverslips onto glass slides using mounting media. Let cure overnight.</li>
	<li>Seal the slides with nail polish and air dry for 1 h.</li>
	<li>Image on confocal microscope with the 60x/1.42 numerical aperture oil immersion lens using the same laser emissions for each sample.</li>
	<li>When analyzing the data, if necessary, make adjustments for brightness and contrast using the same linear adjustment for all samples.</li>
</ol>

<ol>
	<li>Jensen, S., Thomsen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+RNA+viruses:+a+review+of+innate+immune+receptors+involved+in+recognizing+RNA+virus+invasion.">Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion.</a> Journal of Virology. 86, 2900-2910 (2012).</li><li>Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R., Paludan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+produced+by+positive-strand+RNA+viruses+and+DNA+viruses+but+not+in+detectable+amounts+by+negative-strand+RNA+viruses.">Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.</a> Journal of Virology. 80, 6 (2006).</li><li>Triantafilou, K., Vakakis, E., Kar, S., Richer, E., Evans, G. L., Triantafilou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+direct+interaction+of+MDA5+and+the+dsRNA+replicative+intermediate+form+of+positive+strand+RNA+viruses.">Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.</a> Journal of Cell Science. 125, 4761-4769 (2012).</li><li>Onomoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+an+antiviral+stress+granule+containing+RIG-I+and+PKR+in+viral+detection+and+innate+immunity.">Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.</a> PLoS One. 7, e43031 (2012).</li><li>Son, K. N., Liang, Z., Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+detected+by+immunofluorescence+analysis+in+RNA+and+DNA+virus+infections,+including+those+by+negative-sense+RNA+viruses.">Double-stranded RNA is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative-sense RNA viruses.</a> Journal of Virology. 89, 9383-9392 (2015).</li><li>Mateer, E. J., Paessler, S., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+double-stranded+RNA+colocalizing+with+pattern+recognition+receptors+in+arenavirus+infected+cells.">Visualization of double-stranded RNA colocalizing with pattern recognition receptors in arenavirus infected cells.</a> Frontiers Cellular and Infection Microbiology. 8, 251 (2018).</li><li>Buchmeier, M. J., de la Torre, J. C., Peters, C. J., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arenavirdae:+The+viruses+and+their+replication.">Arenavirdae: The viruses and their replication.</a> Fields Virology. 2, (2006).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+interferon+in+Argentine+hemorrhagic+fever.">Endogenous interferon in Argentine hemorrhagic fever.</a> The Journal of Infectious Diseases. 149, 428-433 (1984).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+endogenous+interferon+and+the+clinical+evolution+of+patients+with+Argentine+hemorrhagic+fever.">Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever.</a> Journal of Interferon Research. 5, 383-389 (1985).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+and+old+world+human+arenaviruses+induce+distinct+interferon+response+in+human+cells.">Highly pathogenic new world and old world human arenaviruses induce distinct interferon response in human cells.</a> Journal of Virology. 89, 7079-7088 (2015).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+infection+activates+the+type+I+interferon+pathway+in+a+RIG-I-dependent+manner.">Junin virus infection activates the type I interferon pathway in a RIG-I-dependent manner.</a> PLoS Neglected Tropical Diseases. 6, e1659 (2012).</li><li>Huang, C., Kolokoltsova, O. A., Mateer, E. J., Koma, T., Paessler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+arenavirus+infection+activates+the+pattern+recognition+receptor+protein+kinase+R+without+attenuating+virus+replication+in+human+cells.">Highly pathogenic new world arenavirus infection activates the pattern recognition receptor protein kinase R without attenuating virus replication in human cells.</a> Journal of Virology. 91, 20 (2017).</li><li>Emonet, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+from+cloned+cDNAs+and+in+vivo+characterization+of+recombinant+pathogenic+Romero+and+live-attenuated+Candid#1+strains+of+Junin+virus,+the+causative+agent+of+Argentine+hemorrhagic+fever+disease.">Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid#1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.</a> Journal of Virology. 85 (4), 1473-1483 (2011).</li><li>Child, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+of+the+protein+kinase+R+pathway+in+human+cells+by+Rhesus+Cytomegalovirus.">Antagonism of the protein kinase R pathway in human cells by Rhesus Cytomegalovirus.</a> Journal of Virology. 92, 6 (2018).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li></ol>

The authors have nothing to disclose.

For positive-sense RNA viruses and dsDNA viruses, dsRNA is readily detected with the widely-used J2 anti-dsRNA antibody. However, negative-sense RNA viruses are believed to produce dsRNA at a low or below the detection level using the same antibody2. Thus, many aspects of viral RNA and PRR interaction are largely unclear for negative-sense RNA viruses. We attempted to stain for dsRNA in arenavirus infection using the J2 antibody but the fluorescence signals were not differentiable compared to the mock infection. The Mab 9D5, originally used for pan-enterovirus detection, was found to be specific for dsRNA and more sensitive than the J2 antibody5. This antibody has been used successfully to detect viral dsRNA during negative-sense RNA virus infection, including the prototype arenavirus Lymphocytic choriomeningitis virus5,6,14. Accordingly, we used the Mab 9D5 to co-stain for the presence of dsRNA, the viral NP, and PRRs to further understand their distribution and interaction in individual cells during arenavirus infection.
There are multiple fixation methods that can be used to preserve cell structure. Fixation with MeOH acts by precipitating proteins, whereas paraformaldehyde and formalin crosslinks proteins. MeOH is more effective than aldehydes at conserving the nucleic acids in cells and provides low background immunostaining. Methanol also removes lipids from cells and thus permeabilizes cell membranes at the same time15. In this protocol, the cells are fixed using ice-cold MeOH. Other fixation methods were also attempted, including fixing samples with 4% paraformaldehyde, formalin, paraformaldehyde followed by methanol, and methanol followed by paraformaldehyde. However, high basal level non-specific staining was observed in mock-infected cells when paraformaldehyde or formalin was used. The optimal fixation method is fixation with MeOH at -20 &#176;C based on the sensitivity and specificity of the results. Before primary antibody staining, sample blocking with 3% BSA, 5% BSA, and 10% or 5% goat serum for 30 min to 1 hour was tested, but all resulted in non-specific, background staining. The best results were achieved without the blocking step and directly using 3% BSA in the antibody dilution. The critical steps in minimizing the background signals are the PBS and t-Octylphenoxypolyethoxyethanol washes after fixation. While the commercially available 9D5 antibody is diluted by the vender and ready for direct use, a 1:2 dilution of the antibody with 3% BSA also worked well. In case that the dsRNA signal is weak, the antibody can be used without dilution.
This protocol can be utilized to study the interaction between dsRNA and PRRs in arenavirus infection. While this methodology was developed in a BSL-2 environment, the methanol fixation described herein allows complete inactivation of arenavirus. Therefore, the same protocol can be applied to imaging study for highly pathogenic arenaviruses (i.e., LASV, JUNV and MACV) in a BSL-4 facility. It is also possible to apply this protocol to studies on other RNA virus. To achieve optimal results, a modification of the protocol may be necessary.

The initial step of the induction of the innate immune response is host recognition of double-stranded (ds) RNA by the pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5)1. For positive-sense RNA viruses, dsRNA can usually be readily detected using the J2 or K1 anti-dsRNA monoclonal antibodies (Mab)2. Interaction between dsRNA and PRRs in positive-strand RNA viruses, such as picornavirus, has been characterized using confocal microscopy3. However, for negative-sense RNA virus, visualization and characterization of PRR and dsRNA interaction has been hindered by the lack of sensitive antibodies to dsRNA. RNA fluorescent in situ hybridization (FISH) has been applied to the visualization of viral RNA and PRRs4. Nevertheless, the FISH methodology requires the knowledge of the target RNA sequence and may not be compatible with PRR co-staining. Recently, the 9D5 Mab, which was originally developed for the diagnosis of pan-enterovirus infection, was found to be more sensitive than the J2 Mab and can readily detect dsRNA in negative-sense RNA virus infection5,6. Thus, Mab 9D5 is a novel and useful tool to study viral replication and the interaction between PRR and viral RNA for negative-sense RNA virus.
Arenaviruses are a family of single-stranded, negative-sense RNA viruses, which include several human pathogens, such as Lassa virus (LASV), Jun&#237;n virus (JUNV) and Machupo virus (MACV), that cause severe hemorrhagic fever diseases in humans7. Clinical data from severe and fatal cases of Argentine hemorrhagic fever caused by the New World arenavirus JUNV exhibit unusually high levels of serum IFN-&#945;8,9. We have shown that the pathogenic NW arenaviruses (JUNV and MACV), but not the pathogenic Old World arenavirus, LASV, induce a type I interferon (IFN) response in human monocyte-derived dendritic cells10. Furthermore, RIG-I is one of the sensors mediating type I IFN response in JUNV-infected cells11. We also found that the protein kinase R (PKR) receptor, which is traditionally known for dsRNA recognition, is activated in pathogenic NW arenavirus infection12. To further understand the mechanism of virus-specific IFN response during arenavirus infection, we aimed to develop a protocol to visualize the interaction between viral dsRNA and the cytoplasmic PRRs.
Infection experiments with pathogenic JUNV, MACV and LASV have to be performed in biosafety level 4 (BSL-4) facilities. Thus, in addition to the presumably low level of dsRNA formed in arenavirus infection, meeting the biosafety requirements is another technique challenge when performing imaging studies for these highly pathogenic viruses. By utilizing the 9D5 antibody and the Candid1# vaccine strain of JUNV, a confocal microscopy-based protocol is described in this report, which has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in cells infected by arenavirus in BSL2 labs. The protocol is also suitable for visualization of intracellular distribution of dsRNA and PRR during pathogenic arenavirus infection in BSL4 facilities.

<table><tbody><tr><td>APEX Alexa Fluor 647 antibody labeling kit</td><td>Invitrogen</td><td>A10475</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A4503</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling</td><td>4083</td><td>1:1,000 dilution</td></tr><tr><td>Donkey-anti rabbit Alexa Fluor 594</td><td>Invitrogen</td><td>A-11058</td><td>1:2,000 dilution; Lot #: 1454437</td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Bio</td><td>S11150</td><td></td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>Goat-anti mouse Alexa Fluor 488</td><td>Invitrogen</td><td>A-11029</td><td>1:2000 dilution; Lot #: 1874804</td></tr><tr><td>Human lung epithelial A549 cells</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>A412</td><td>Stored at -20 &amp;deg;C</td></tr><tr><td>Mouse MAb anti-JUNV NP</td><td>BEI</td><td>NA05-AG12</td><td>Conjugated to Alexa-647 at 1:1,000 dilution</td></tr><tr><td>Mouse MAb pan-Enterovirus 9D5 Reagent</td><td>Millipore Sigma</td><td>3361</td><td>ready for use; diluted to 1:2; Lot #: 3067445</td></tr><tr><td>PBS supplemented with Ca and Mg</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>PDL Coated coverslips</td><td>Neuvitro</td><td>H-12-1.5-pdl</td><td></td></tr><tr><td>ProLong Gold antifade</td><td>Invitrogen</td><td>P10144</td><td></td></tr><tr><td>Rabbit MAb MDA-5</td><td>Abcam</td><td>ab126630</td><td>1:250 dilution; Lot #: GR97758-7</td></tr><tr><td>recombiant Candid#1 strain of JUNV</td><td>Lab generated</td><td>Lab generated</td><td>As previously described in reference 13.</td></tr><tr><td>RIG-I mouse MAb conjugated to Alexa-594</td><td>Santa Cruz</td><td>sc-376845</td><td>1:1000 dilution; Lot #: AO218</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787&amp;nbsp;</td><td></td></tr></tbody></table>

confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

This protocol was applied to study the distribution and colocalization between the RLRs (RIG-I and MDA-5) and dsRNA in JUNV-infected cells. As shown in Figure 1 and Figure 2, the accumulation of dsRNA increases over time as viral infection progresses. Concentrated MDA-5 (Figure 1) and RIG-I (Figure 2) signals were found colocalized with the punctate structures of the NP and dsRNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59095/59095fig1.jpg" /> 
Figure 1: Time course of dsRNA and JUNV NP formation and the distribution of MDA-5. JUNV-infected and mock-infected A549 cells were fixed, stained and imaged according to the protocol at 24, 36, and 48 hours post infection (HPI). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59095/59095fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59095/59095fig2.jpg" /> 
Figure 2: Time course of dsRNA and JUNV NP formation and the distribution of RIG-I. JUNV-infected and mock-infected A549 cells were fixed, stained and imaged according to the protocol at 24, 36, and 48 HPI. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59095/59095fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

confocal-imaging-double-stranded-rna-pattern-recognition-receptors

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.6K Views.  University of Texas Medical Branch. Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Video: Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Genetics

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biology

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

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Chapters in this video

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning