The type I interferon (IFN) response is a transient innate immune defense system that, upon 19 activation by viral infection or therapeutic IFN treatment, induces the transcription of hundreds of 20 interferon-stimulated genes (ISGs) (1). Many ISGs have characterized antiviral roles that restrict 21 viral replication by either interfering with viral processes directly or altering cellular pathways 22 required for viral replication (2). However, because replication mechanisms and points of 23 interaction with the cell differ between viruses, individual ISGs have varying potencies against 24 different viruses (3-5). As a result, unique combinations of ISGs are thought to mediate successful 25 antiviral responses against distinct viruses (1, 6). 26
The canonical activation pathway of the type I IFN signaling pathway induced by viral infection, 28 or after therapeutic administration (7), is well understood (8, 9) . Extracellular IFN is bound by its 29 cognate plasma membrane-localized receptor (IFNAR1/2). Downstream effectors (JAK proteins) 30 are phosphorylated to then activate formation of the interferon-stimulated gene factor 3 (ISGF3) 31 complex. Finally, the ISGF3 complex of STAT1, STAT2, and IRF9 translocates to the nucleus (8) 32
and binds the interferon sensitive response element (ISRE), with the consensus DNA motif 33 GAAANNGAAA, to activate transcription of ISGs (10). 34 35 As infection is cleared and virally-derived innate immune activators become scarce, interferon 36 production is reduced and the interferon-stimulated gene response is downregulated. To facilitate 37 this return to cell homeostasis, IFN induced negative regulators, such as SOCS1 (11) and USP18 38 transduced cells, and cells that were nonresponsive to type I IFN. Raw sequencing data was 96 aligned, mapped, and subsequently analyzed using the MAGeCK pipeline (29) to generate z-score 97 values for each gene. Genes were defined as "hits" if their z-scores exceeded two standard 98 deviations from the mean, resulting in an overlap of 10 high-confidence genes between the two 99 screen replicates (Fig. S1A, Table S1 , Data files S1 and S2). We were seeking to identify 100 regulators of the IFN response that are regulated by IFN themselves; therefore, we selected hits 101 for validation previously reported to have at least a two-fold induction after IFN stimulation in the 102 Interferome database (30). This analysis identified three hits (C1GALT1, ETV7, and NUP153) as 103 potential negative regulators of the type I IFN response (Table S1) . 104
To validate our three hits, and to eliminate any potential off-target effects of CRISPRa, we cloned 106 the three ORFs and validated overexpression of the genes in 293T cells (Fig. S1B) . Co-transfection 107 of the overexpression plasmids and IFN response plasmid, followed by stimulation with IFN-a, 108 resulted in lower reporter expression compared to a control mCherry-expressing plasmid (Fig. 1E  109   Fig. 1. (continued) untreated control). (D) Diagram of CRISPRa screen workflow to identify negative regulators of the type I IFN response. (E) Flow cytometry plots of 293T cells transfected with the IFN response reporter and overexpression plasmids for the indicated screen hits and then treated with IFN-α (100 U/mL, 9 h). (F) Quantification of E showing brightness of cells expressing GFP compared to the mCherry-expressing control (data shown as mean ± SD, n=4). (G) Brightness of cells expressing GFP from a constitutively expressing plasmid in cells overexpressing the indicated genes (positive control = EIF2AK1/HRI, shuts off translation) compared to control (data shown as mean ± SD, n=4). (H) Western blot showing ETV7 protein levels in 293T cells transfected with the ETV7 overexpression plasmid. Stain-free gel imaging was used to confirm equal loading. (I) Endogenous ISG mRNA expression levels measured using RT-qPCR after IFNα treatment (100 U/mL, 9 h) (data shown as mean ± SD, n=4). (J) Western blot comparing IFIT1 protein levels in control and ETV7 overexpressing cells after IFN-α treatment (500 U/mL, 18 h). Stain-free gel imaging was used to confirm equal loading. For all panels: P-values calculated using unpaired, two-tailed Student's t-tests (*p<0.05, **p<0.001) compared to mCherry-expressing control samples unless otherwise noted. Diagram showing the ETV7 protein domains and amino acid changes made to generate the DNA binding mutant, ETV7(KALK). (B) Western blot showing ETV7 protein levels in 293T cells transfected with WT or DNA binding mutant (KALK) ETV7 expression plasmids. Stain-free gel imaging was used to confirm equal loading. (C) Percent of 293T cells expressing GFP from the IFN response reporter with overexpression of WT or DNA binding mutant (KALK) ETV7 after IFN-α treatment (100 U/mL, 9 h) compared to control (data shown as mean ± SD, n=4). (D) ETV7's DNA binding position weighted matrix (PWM) generated using enoLOGOS (99) with data from Wei et al. (36) . (E) Diagrams of the IFN response reporters containing (+ETS) and not containing (-ETS) potential ETV7 binding sites (ETS site, highlighted in yellow). (F) Normalized percent of 293T cells expressing GFP from IFN response reporters either containing or not containing ETS sites after overexpression of ETV7 and IFN-α treatment (100 U/mL, 6 h) compared to mCherry-expressing control (data shown as mean ± SD, n=4). (G) Diagram of the DNA pull-down experiment using biotinylated DNA and streptavidin-coated magnetic beads to show binding of a transcription factor to a specific DNA sequence. Sequences of biotinylated DNA with IFN-a had no measurable effect on expression of the IFN response reporter, in contrast to 135 WT ETV7 overexpression (Fig. 2C, Fig. S2A ). 136 137 ETV7 has been reported to bind the canonical ETS family DNA motif, GGAA (36), known as an 138 "ETS" site ( Fig. 2D) . The original IFN response reporter design contained multiple ETS sites (Fig.  139 2E), which likely explains why it is negatively impacted by ETV7 in our screen. To test the 140 requirement of ETS sites for ETV7 repressive activity against our reporter, we generated an IFN 141 response reporter containing seven consensus ISREs from canonical ISGs that all lack ETS sites 142 (ISRE -ETS) (Fig. 2E) . We transfected the two reporter plasmids (ISRE +ETS and ISRE -ETS) 143 independently into 293T cells and stimulated with IFN-a. As expected, both reporter plasmids 144 responded to IFN treatment and were repressed by overexpression of known negative regulators 145 of the type I IFN response (SOCS1, USP18) that function upstream of transcription (Fig. S2B) . 146
When ETV7 was transfected into cells with the IFN response reporters and stimulated with IFN 147 however, the repressive activity of ETV7 was restricted to the reporter plasmid containing ETS 148 motifs (Fig. 2F, Fig. S2C) . 149 150 Fig. 2. (continued) containing ETS sites after overexpression of ETV7 and IFN-α treatment (100 U/mL, 6 h) compared to mCherry-expressing control (data shown as mean ± SD, n=4). (G) Diagram of the DNA pull-down experiment using biotinylated DNA and streptavidin-coated magnetic beads to show binding of a transcription factor to a specific DNA sequence. Sequences of biotinylated DNA bait used to show binding of ETV7 to the WT ISG15 ISRE (93) and loss of binding with mutation of the ETS binding site (-ETS). (H) Western blot of DNA pull-down using biotinylated oligos containing the ISG15 ISRE sequence in its wild-type form (WT) or with a single nucleotide mutation (-ETS) to eliminate the ETS site, incubated with nuclear lysate from cells expressing ETV7 with IFN treatment (100 U/ml, 9 h). (I) Heat map displaying RNA levels of genes upregulated at least 2.5-fold following IFN-α treatment (100 U/mL, 9 h) in control (mCherry) cells. Yellow = upregulated, blue = downregulated. For all panels: P-values calculated using unpaired, two-tailed Student's t-tests (*p<0.05, **p<0.001) relative to IFN-stimulated, mCherry-expressing control samples.
To provide evidence of ETV7 directly binding ISRE motifs containing ETS sites, we performed a 151 DNA oligo-based pull-down experiment using the ISG15 ISRE sequence (Fig. 2G) . We chose this 152 ISRE because 1) it contains an ETS binding site, 2) it was included in our initial IFN response 153 reporter, and 3) ETV7 was shown to impact ISG15 induction (Fig. 1I) . In addition to the wild-154 type ISG15 ISRE biotinylated oligos, we tested biotinylated oligos with a single nucleotide 155 mutation that eliminates the ETS site in the ISG15 ISRE (-ETS). We generated nuclear lysates 156 from 293T cells overexpressing ETV7 and treated with IFN-a, incubated the lysate with either the 157 biotinylated WT or -ETS ISG15 ISRE oligos, and pulled down the biotinylated DNA using 158 streptavidin-coated magnetic beads. Western blotting for ETV7 protein revealed the transcription 159 factor bound the WT ISG15 ISRE containing the ETS site, but not the mutated version of the 160 promoter lacking the ETS site (Fig. 2H) . Together, these experiments demonstrate that the 161 repressive activity of ETV7 requires both its ability to bind DNA and the presence of ETS sites in 162 the target ISG promoters. 163
Since consensus ISREs can either contain or lack a GGAA motif (Table S2) (Table S3) ; this is expected, as ETV7 is an ETS family member and the entire family binds 176 the same core motif, GGAA. In contrast, the genes specifically downregulated by ETV7 during 177 IFN treatment were highly enriched for processes and pathways associated with responses to 178 interferon and viral infection (Table S4) . To visualize the effect of ETV7 upregulation specifically 179 on ISGs, we generated a heat map of the genes induced at least 2.5-fold upon IFN treatment in our 180 RNA sequencing experiment (Fig. 2I) . Consistent with our hypothesis, we found some genes were 181 suppressed by ETV7 overexpression more than others (Fig. 2I) . 182 183 Although we had previously immunoprecipitated ETV7 with ISRE-containing oligos (Fig. 2H) , 184 we next wanted to demonstrate ETV7 occupancy on native ISG promoters for genes targeted by 185 ETV7 via chromatin immune-precipitation followed by quantitative PCR (ChIP-qPCR). To enable 186 these experiments, we first generated a FLAG-tagged ETV7 (Fig. S2E) and demonstrated that the 187 tag does not disrupt its ability to repress the IFN response reporter (Fig. S2F) . We next selected 188 the promoter of IFI44L for ChIP analysis as it was both highly affected by ETV7 and also harbors 189 many ETS sites within ISRE-like sequences in its promoter ( Fig. 2I and Fig. S2G and H) . To 190 perform ChIP-qPCR, we generated sheared chromatin after cross-linking cells transfected with 191 FLAG-tagged ETV7 and treated with IFN-a. We then performed immunoprecipitation using either 192 nonspecific IgG or anti-FLAG antibodies raised in mice or rabbits. After qPCR analysis, we found 193 no enrichment for a negative control region (the gene desert on chromosome 12) between the IgG 194 and anti-FLAG samples (Fig. S2I) . In contrast, DNA corresponding to the IFI44L promoter was 195 significantly enriched with both the rabbit and mouse anti-FLAG samples compared to the 196 nonspecific IgG samples (Fig. S2I) . These experiments demonstrate that ETV7 acts as a 197 transcription factor to bind and suppress ISG promoters that containing GGAA motifs. Our experiments up to this point had primarily utilized overexpression of ETV7. One major 202 disadvantage of this approach is the non-physiological kinetics and magnitude of ETV7 expression 203 relative to what is observed after IFN stimulation in cells (Fig. 3A) . To define the physiological 204 effects of ETV7 induction during the IFN response, we performed a series of loss-of-function 205 experiments. Our expectation was that the knockout of ETV7 would have the reciprocal effect on 206 IFN responses as protein overexpression (38). We transduced A549-IFN response reporter cells 207
(the original reporter with ISRE +ETS sites) with Cas9 and separately, two independent sgRNAs 208 targeting ETV7, KO1(sg16667) and KO2(sg16668), generated pooled lines using antibiotic 209 selection, and then stimulated with IFN-a. Both guides resulted in significantly more IFN-induced 210 sfGFP expression compared to a control sgRNA (Fig. 3B, Fig. S3A) . 211 212 We next generated clonal ETV7 knockout A549 lung epithelial cell lines (KO1-1, KO1-2) lacking 213 the reporter and sequenced the resulting DNA lesions to confirm ETV7 knockout. Since ETV7 is 214 normally only expressed after IFN stimulation, we treated with IFN-a and verified a reduction in 215 ETV7 expression at the RNA level, presumably via nonsense mediated RNA decay (Fig. 3C) . A 216 representative housekeeping gene (ACTB) displayed no significant increases in transcription in 217 any of the ETV7 KO clones (Fig. 3D) . We then selected twelve ISGs (IFITM1, RSAD2, OAS1-218 3, IFIT1-3, IFI44L, ISG15, BST2, and IFI44) that were highly impacted by ETV7 in our RNA 219 sequencing results for RT-qPCR analysis after IFN treatment. Compared to clonal lines containing 220 a non-targeting guide, the ETV7 KO lines showed significant increases in induction of each of the 221 twelve ISGs (Fig. 3E-K, Fig. S3B-F) 
In trying to predict the physiological significance of excessive induction of these ETV7-regulated 228
ISGs during the type I IFN response, we noted that many have recognized antiviral functions (2, 229 (B) Percentage of cells expressing GFP from the IFN response reporter in two A549 ETV7 KO cell lines (pooled, 2 different guides) after IFN-α treatment (1000 U/mL, 6 h) compared to nontargeting control cells (data shown as mean ± SD, n=3). (C) mRNA levels of ETV7 in nontargeting control and ETV7 KO A549 clonal cell lines after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). (D-K) mRNA levels of a housekeeping gene (D) and ISGs (E-K) in control and ETV7 KO A549 clonal cell lines after IFN-α treatment (100 U/mL, 9 h) (data shown as mean ± SD, n=4). For all panels: P-values calculated using unpaired, two-tailed Student's t-tests (*p<0.05, **p<0.001) compared to IFN-stimulated, non-targeting sgRNA control samples. compared to control cells (Fig. 4A) . This was the anticipated outcome because loss of a negative 242 regulator (i.e. ETV7) is expected to enhance expression of antiviral ISGs. We also measured 243 infectious viral titers and found a significant reduction in our ETV7 KO cells compared to control 244 cells (Fig. S4A) . Using a fluorescent reporter strain of PR8 (PR8-mNeon) (48), we next visualized 245 infection and spread. As expected, we observed fewer cells expressing mNeon in ETV7 KO cells 246 using both microscopy (Fig. 4B) and flow cytometry readouts (Fig. 4C) . We also tested whether 247 this phenotype would extend to a more contemporary H1N1 IAV strain, control cells (Fig. 4D and E) . In order to rule out non-IFN-related effects of ETV7 on inhibition 252 of influenza viral replication, we also performed these experiments with a non-influenza virus. We Ebola virus (57), SARS-CoV-2 (58-60)). Therefore, we wanted to assess whether suppressing 266 ETV7 could be an effective strategy to enhance the antiviral effects of therapeutic IFN 267 administration. In support of this idea, we found treatment of ETV7 KO cells with IFN-a prior to 268 infection with PR8-mNeon led to an additional 5-fold enhancement of viral restriction compared 269 to IFN-treated WT control cells (Fig. 4G) 
to a control cell line after pre-treatment with IFN-α (100 U/ml, 6 h) then infection with PR8-mNeon reporter virus (24 h, MOI=0.1, multicycle) (data shown as mean ± SD, n=4). (H) qPCR detecting SARS-CoV-2 N vRNA in infected cells from ETV7 KO A549 clonal cell lines compared to a control cell line after pre-treatment with IFN-α (1000 U/ml, 6 h) then infection with SARS-CoV-2 virus (24 h, MOI=0.1) (data shown as mean ± SD, n=4). For all panels: P-values calculated using unpaired, two-tailed Student's t-tests (*p<0.05, **p<0.001) compared to infected, nontargeting sgRNA control samples. experiment, we found that pre-treatment of ETV7 KO cells with IFN-a resulted in a further 10-274 fold reduction in SARS-CoV-2 vRNA compared to IFN-treated control cells (Fig. 4H) . This 275 enhanced control was likely mediated via the ISG LY6E, which potently restricts SARS-CoV-2 276 (67, 68) and we found to be regulated by ETV7 (Fig. S4B) In this study, we performed a CRISPR activation screen to identify negative regulators of the type 282 I IFN response. Specifically, we were interested in negative regulators that contribute to the types 283 of differentiated ISG profiles that are essential for effective control of viral infections. From this 284 screen, we identified ETV7 as a negative IFN regulator and subsequently showed it acts as a 285 transcription factor to repress subsets of ISGs through its recognized DNA binding motif. We also 286 demonstrated that the regulatory activity of ETV7 impacts the replication and spread of multiple 287 strains of influenza viruses. ETV7 loss also increased the antiviral effects of exogenous interferon 288 treatment against an H1N1 influenza virus and SARS-CoV-2. These findings demonstrate the 289 importance of ETV7 in fine-tuning the IFN response through specificity and transcriptional 290 repression to regulate antiviral ISG targets, and its potential as a target to enhance antiviral IFN-291 based therapeutics. 292 293 ETV7 is a member of the ETS family of transcription factors. This family performs diverse 294 functions despite recognizing the same core DNA sequence, GGAA. Because these factors share 295 the same core motif, specificities are gained in other ways such as expression patterns (cell type, 296 basal expression, or immune pathway induction), active or repressive activity, and potential 297 binding partners (69, 70). ETV7 is induced as an ISG and is repressive, whereas most ETS 298 transcription factors are activators, including ETS transcription factors recognized to assist in the 299 induction of ISG expression (e.g. ELF1 (25), PU.1 (71)). We also found repeatedly in our work 300 that the reporters and genes impacted by ETV7 contained ETS sites within ISRE sequences (Fig.  301   2E-H, S2G-I, S4B) . This is not unexpected because it is recognized that many ETS family 302 members bind extended motifs similar to ISREs called ETS-IRF composite elements (EICE) (72). 303 EICE-associated activity is reported to require an IRF binding partner to direct ETS transcription 304 factor activity (71, 73); therefore, it is likely ETV7 has an IRF binding partner. If ETV7 does 305 require a binding partner, this protein's induction and distribution likely contribute to the timing, 306 gene targets, and activity of ETV7 during the IFN response. It is known that IRFs can be basally (1, 6) . We compared the induction of ETV7 with IFI44L, IFIT1, 317 ISG15 and RSAD2, and we observed ETV7 is upregulated at earlier time points than these 318 prototypical ISGs (Fig. 3A) . We expanded our analysis to published datasets of human gene 319 expression during respiratory infections and concluded that ETV7 is generally induced earlier than 320 many ISGs (75). Although not the focus of our study, ETV7's early induction pattern suggests it 321 may be a key regulator of the first stages of IFN-mediated gene induction. We favor a model 322 20 wherein early ETV7 expression is responsible for reducing the accumulation, or delaying the 323 expression, of ISGs controlled by ISREs and promoters containing ETS motifs. 324 325 ETV7 is induced during infections across many vertebrate species (76, 77) , indicating a potential 326 conserved, relevant role in the immune response; however, ETV7 has been lost in mice and closely 327 related rodents (78). Since mice and rodents have an intact interferon response pathway, a natural 328 question is: how are the activities of ETV7 being accounted for in these animals? While we have 329 no clear answer from the data in this study, it is well-recognized that IFN responses contain many 330 redundancies (41). Accordingly, we believe other ETS family members, potentially the closely 331 related ETV6 (which is also induced by IFNs), may perform the role of ETV7 in mice (79). Future 332 studies will be required to test the hypothesis that mice induce an ETV7-related alternative during 333 the type I IFN response. 334 335 Another important question is why the IFN-induced activity of ETV7 has been maintained 336 throughout evolution. In this report, we provide evidence that ETV7's activity reduces a cell's 337 ability to restrict virus infection; this seems counterintuitive to ETV7 benefitting the host. We 338 hypothesize that regulators like ETV7 are important to prevent excessive inflammatory signaling. 339
It is appreciated that negative regulators of the IFN response are required to prevent extreme and 340 prolonged immune responses, which are associated with poor disease outcomes after infection 341 (80-82). ETV7 potentially contributes to the cumulative activities of negative IFN regulators to 342 limit IFN responses during pathogen clearance. Additionally, it stands to reason that individual 343
ISGs have different toxic effects on the cell. It is tempting to speculate that ETV7 suppresses ISGs 344 whose accumulation is particularly harmful to cell viability and host recovery after infection. In conclusion, this study identified ETV7 as a negative regulator of the type I IFN response. 360
Previously, ETV7 was appreciated to be an ISG; however, a specific function during the IFN 361 response was unknown. We determined that ETV7 acts as a transcription factor to target specific 362 ISGs for repression, potentially contributing to the complex ISG transcriptional landscape. 363
Additionally, many of the ETV7-regulated ISGs restrict respiratory viruses (42, 67, 68), and we 364 showed that loss of ETV7 can further enhance the ability of type I IFN to control influenza virus 365 and SARS-CoV-2 replication. Further work is required to understand the complexity of IFN 366 regulation, while therapeutic targeting of factors like ETV7 could lead to the development of a 367 new class of host-directed antivirals that can enhance or tailor ISG responses to specific viruses. 368
Cloning 370
To generate reporters sensitive to IFN, we designed gBlocks (IDT) containing ISREs to be cloned 371 into the pTRIP vector ahead of a minimal CMV promoter controlling expression of sfGFP. To 372 clone and express the open reading frames (ORFs) of our screen hits, we designed primers for 373 cloning into the pLEX-MCS vector using Gibson Assembly (NEB). To amplify ETV7, NUP153 374 and USP18 we used cDNA templates from Transomic Technologies. To amplify C1GALT1 and 375 EIF2AK1 we used RNA from IFN-stimulated A549 cells. The DNA binding mutant ETV7, 376 ETV7(KALK) (35), and codon-optimized SOCS1 expression plasmids were generated using a 377 gBlock (IDT). FLAG-tagged ETV7, ETV7(FLAG), was generated using a primer including the 378 FLAG tag. Non-targeting and ETV7-targeting CRISPR KO sgRNAs were cloned by annealing 379 oligos encoding the desired sgRNA sequence and ligating them directly into the lentiCRISPRv2 380 vector (Addgene). DNA was transformed into NEB 5-alpha high efficiency competent cells. Insert 381 size was verified with PCR and purified plasmids were sequenced using Sanger sequencing. Cells were trypsinized and 1x10 6 cells were pelleted at 800 x g for 5 min. Equal amounts of cellular 456 material were loaded into 4-20% acrylamide gels (Bio-Rad) and imaged using a ChemiDoc 457
Imaging System (Bio-Rad). Protein was transferred to a nitrocellulose membrane at 60V for 60 458 min. PBS with 5% (w/v) non-fat dried milk and 0.1% Tween-20 were used to block for 1 h at 4°C. 459
Primary antibodies were then incubated with the membrane overnight at 4°C. Antibodies used 460 were rabbit anti-ETV7 (Sigma, HPA029033), rabbit anti-IFIT1 (Cell Signaling, D2X9Z), mouse 461 anti-FLAG (Sigma, F3165) and mouse anti-atubulin (Sigma, T5168). Membranes were washed 462 five times in PBS with 0.1% Tween-20 and then an anti-rabbit-HRP (Thermo, A16104) or anti-463 mouse-HRP (Thermo, A16072) secondary antibody was added for 1 h. The membrane was then 464 washed five times and Clarity or Clarity Max ECL substrate (Bio-Rad) was added before being 465 Motif), rabbit anti-FLAG (Cell Signaling, 14793S), and mouse anti-FLAG (Sigma, F3165). 513
Quantitative PCR was performed using SYBR Green (Bio-Rad) and primers targeting the Chr12 514 gene desert (Active Motif) or IFI44L promoter (forward primer = 5' 515 TTTCATGCCTGCCTACATAC 3', reverse primer = 5' ATGCCAACTGCCACTAAC 3') in a 516 region containing two potential ISRE sequences overlapping with ETS sites, and analyzed using 517 the ChIP-IT qPCR Analysis kit (Active Motif). at 4°C for 2-3 h before scoring. Infectious viral titers were determined using standard plaque assay 538 procedures on MDCK cells. Infected cell supernatants were collected at 18 h, serially diluted, and 539 used to infect confluent 6-well plates for 1 h before removing the virus and adding the agar overlay. 540
Cells were then incubated at 37°C for 48 h before being fixed in 4% PFA overnight. The 4% PFA 541 was then aspirated, and the agar layer was removed before washing cells with PBS. Serum from 542 WT PR8 infected mice was diluted 1:2,000 in antibody dilution buffer (5% (w/v) non-fat dried 543 milk and 0.05% Tween-20 in PBS) and incubated on cells at 4°C overnight. Cells were then 544 washed twice with PBS and incubated for 1 h with anti-mouse IgG horseradish peroxidase (HRP)-545 conjugated sheep antibody (GE Healthcare) diluted 1:4,000 in antibody dilution buffer. Assays 546 were then washed twice with PBS and exposed to 0.5 mL of TrueBlue peroxidase substrate (KPL) 547 for 20 min. Plates were then washed with water and dried before plaques were counted. 548 549 550 