Construction and rescue of Min AL

The new CPD virus Min AL was created by combining the CPD ORFs of Min A (CPD NS1, NS2, N, P, M, SH) and Min L (CPD L) (Fig 1A). Thus, Min AL contains CPD versions of seven of the 11 RSV ORFs, with a total of 2,073 silent mutations within ORFs. This represents a 3- and 1.5-fold increase over Min A (695 silent mutations) and Min L (1,378 silent mutations; Fig 1A), respectively. The full-length genome of wt RSV contained only 87 CpGs and 1,462 UpAs, while that of Min AL contained 426 CpGs and 1,770 UpAs. When the analysis was focused on the combined ORFs, wt RSV contains 79 CpGs and 1,312 UpAs, Min A contains 205 CpGs and 1,418 UpAs, Min L contains 292 CpGs and 1,514 UpAs, and Min AL contains 418 CpGs and 1,620 UpAs (S1 Table). Min AL was readily rescued by reverse genetics as previously described [33] and passaged once to prepare working stocks. Illumina whole-genome deep-sequencing of Min AL stocks confirmed that the recovered genomes were free of any adventitious mutations with an abundance of ≥1% of reads.

Min AL combines temperature sensitivity from Min A and Min L

We first evaluated the replication of Min AL in comparison to the previous CPD viruses in multi-cycle in vitro growth kinetics experiments (Fig 1B). Vero cells were infected with Min A, Min L, Min AL, Min FLC or wt RSV using an multiplicity of infection (MOI) of 0.01 plaque forming units (pfu)/cell. Infected cells were incubated at 32°C, a permissive temperature for temperature-sensitive mutants that also simulates the temperature of the upper respiratory tract, or the more-restrictive temperature of 37°C that simulates the temperature of the lower respiratory tract and core body. Virus replication was evaluated for 12 and 10 days, respectively.

At 32°C (Fig 1B, left panel), wt RSV replicated efficiently, reaching 7.4 log₁₀ pfu/ml by day 6 post-infection (pi), as is typically observed. Replication of all CPD RSVs was reduced and delayed, with replication of Min FLC being the most affected, reaching a maximum peak titer of 6.0 log₁₀ pfu/ml by day 12 pi, similar to our previous results [33]. Replication of the new Min AL virus was comparable to Min L, and slightly more efficient than Min A, reaching a peak titer of 6.7 log₁₀ pfu/ml on day 12 pi. At 37°C (Fig 1B, right panel), wt RSV replicated efficiently and reached a peak titer on day 3 pi (7.2 log₁₀ pfu/ml). As previously described [33], replication of Min A and Min L was strongly reduced (by 48-fold and 1000-fold, respectively) and only a baseline replication of Min FLC was detected. The replication of Min AL was reduced compared to Min L by about 60-fold, indicating that the new virus was more temperature sensitive than Min A and Min L.

The plaque sizes of wt and CPD RSVs at 32°C on Vero cells were evaluated following incubation for seven days under a methylcellulose overlay (Fig 1C). Compared to wt RSV, the plaque sizes of all CPD RSVs were significantly reduced by 3- to 5-fold (p<0.0001). The plaques formed by Min AL appeared to be slightly smaller than Min L, slightly larger than Min A, and even larger than Min FLC, but the differences were not significant.

We next determined the shut off temperature (T_SH) for plaque formation by Min AL, defined as the lowest temperature at which there is a reduction in plaque number compared to 32°C that is 100-fold or greater than that observed for wt RSV between the two temperatures (S2 Table). Wt RSV efficiently formed plaques at 40°C as typically observed, while the T_SH of Min A, Min L and Min FLC were 40°C, 38°C and 36°C, respectively, as previously described [33]. The T_SH of Min AL was found to be 36°C, suggesting that its temperature sensitivity phenotype had contributions from both Min A and Min L.

Genetic and phenotypic stability of Min AL when serially passaged at increasing temperature (in vitro stress test)

Since Min AL exhibited a strong temperature sensitivity, we evaluated the genetic stability and the stability of the temperature-sensitivity phenotype of Min AL in an in vitro stress test (Fig 2). Twelve 25 cm² flasks of Vero cells were inoculated with Min AL using an initial MOI of 0.1 pfu/cell. Each flask represented an independent lineage. Ten lineages were passaged in parallel starting at the permissive temperature of 32°C, with an increase in temperature of 1°C following every second passage up to 40°C, for a total of 18 passages representing approximately 4 months of continuous culture. In parallel, two additional lineages were passaged 18 times at 32°C as controls. For each passage, cell monolayers from each lineage were harvested when the cytopathic effect was extensive, typically between 4 and 7 days after infection, and one-fifth of the clarified culture medium was used to infect a fresh monolayer of Vero cells. The remainder of the clarified medium was snap frozen in aliquots on dry ice for subsequent titration and, for supernatants collected after 14 passages at increasing temperatures or after 18 passages for the controls, for whole-genome Illumina deep sequencing.

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Fig 2. In vitro stress test of Min AL.

Twelve replicate monolayer cultures of Vero cells in 25 cm² flasks were inoculated at an MOI of 0.1 pfu/cell with Min AL and serially passaged in parallel. Each flask represented a separate lineage. Two lineages (A) were passaged 18 times at the permissive temperature of 32°C as controls. For the other ten lineages (B), the temperature of incubation was increased by one°C after every other passage from 32°C to 40°C for a total of 18 passages. When extensive syncytia were observed or when cells started to detach (typically between six and 11 days pi), cells were scraped into the medium and the supernatants were harvested and clarified by low-speed centrifugation. One ml of the total of five clarified ml was used to inoculate the following passage. The remaining supernatants were aliquoted and snap frozen in dry ice for subsequent titration at 32°C and sequence analysis. The passage number and corresponding temperatures of incubation are indicated, as well as the virus titer of the initial inoculum and passage harvests. Each lineage is represented separately by a different symbol. The passages incubated at the 36°C shut-off temperature of Min AL (T_SH, P9 and 10) are indicated with a black arrow. Red arrows mark supernatants collected after 14 passages at increasing temperatures or 18 passages at 32°C for the controls that were used for viral RNA extraction and sequence analysis.

https://doi.org/10.1371/journal.ppat.1012198.g002

For the two control lineages of Min AL passaged at 32°C, the virus replicated efficiently for all 18 passages, reaching titers that varied between 5.5 and 7.0 log₁₀ pfu/ml (Fig 2A). Virus titers of each of the ten Min AL lineages passaged at increasing temperatures increased by 3-fold during the first two passages at 32°C, reaching on average 6.9 log₁₀ pfu/ml, similar to the control lineages (Fig 2B). From P3 to P8 (33°C to 35°C), titers in each lineage decreased steadily by about 30-fold on average to 5.5 log₁₀ pfu/ml. Then, from P9 to P14 (first passage at 36°C to second passage at 38°C), replication was variable among the lineages. Titers in six of the 10 lineages continued to decrease between 6- and 1,800-fold between P9 to P14; the titers in one lineage remained unchanged from P9 to P14; and titers in two lineages increased by 2- to 4-fold from P9 to P14. From P15 to P16 (the two passages at 39°C), titers sharply decreased in all lineages, ranging from 4.1 log₁₀ pfu/ml to below the limit of detection. Finally, at P17 and P18 (the two passages at 40°C), replication of all Min AL lineages was below the limit of detection. The observation that most of the lineages replicated efficiently beyond the T_SH for Min AL of 36°C, but that none of them replicated at 40°C, suggested that they had a partial loss of the ts phenotype.

Min AL lineages acquired multiple, different point mutations in both CPD and wt ORFs during the in vitro stress test

To identify mutations that might be responsible for the partial loss of the ts phenotype during the in vitro stress test, viral RNA was extracted from clarified cell culture supernatants harvested at the end of P14 (second passage at 38°C), corresponding to the last passage at which replication was still detected in most of the lineages (Fig 2B, red arrow). Viral RNA also was extracted from supernatants from the two control lineages at P18. The viral RNAs were amplified by overlapping RT-PCRs using RSV-specific primers. PCR amplicons covering the whole genome, with the exception of the outer-most primers at the 3’- and 5’-ends (nucleotides 1–23 and 15,174–15,223), were sequenced by Illumina deep sequencing. PCR amplicons were not obtained for P14 of lineages #2 and #4, presumably due to low viral titers, and thus these two lineages were not evaluated further.

Sequence analysis of these eight “stressed” lineages (#1, 3, 5, 6, 7, 8, 9 and 10) identified a total of 38 different prominent (>30% of the sequencing reads) point mutations (Table 1). Of these 38 different mutations, three were present in two lineages each and one in three lineages; the remaining 34 mutations were present in only one lineage each. Each lineage had 3–8 mutations with >30% abundance.

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Table 1. Mutations detected at a frequency of >30% in eight of the ten Min AL lineages at the end of passage 14 (2^nd passage at 38°C) as well as in each of the two controls at passage 18 (32°C).

https://doi.org/10.1371/journal.ppat.1012198.t001

Of the 38 prominent mutations from the stressed lineages, only three were in non-protein-coding regions: namely, in the P gene-start signal (c2334a, the 5^th nucleotide of the signal GGGGCAAAT, found in two lineages), the 5’ untranslated region (UTR) of the P gene (a2344g), and the L gene-start signal (a8494g, the 4^th nucleotide of the signal GGGACAAAAT). The remaining 35 mutations (92%) were in ORFs; of these, 25 (71%) were missense mutations, suggesting a selective pressure for aa change. Of these 25 missense mutations, four (16%) were in NS1, three (12%) in N, four (16%) in P, one (4%) in F, 11 (44%) in M2-1, one (4%) in M2-2 and one (4%) in L. Thus, Min AL lineages passaged under increasing temperature acquired missense mutations in the CPD NS1, N, P and L ORFs but also in the F, M2-1 and M2-2 ORFs which had not been subjected to CPD, with about half of the missense mutations found in M2-1.

After 18 passages of the two control lineages at the permissive temperature of 32°C, only a single point mutation of >30% abundance was identified, occurring in one of the two lineages (Table 1, Ct1). This mutation was located in the P gene-start signal (a2337t at the 8^th nucleotide of the signal GGGGCAAAT). Thus, Min AL was genetically stable at the permissive temperature.

Reintroduction of mutations identified at P14 of the in vitro stress test (second passage at 38°C) into the Min AL backbone

We next attempted to identify the mutations responsible for the partial loss of the ts phenotype of Min AL during the in vitro stress test (Table 1). To do so, we considered four lineages (#1, 3, 6 and 7, Table 1) that replicated the most efficiently at P14 (titers >5.0 log₁₀ pfu/ml) and thus might be expected to contain mutations with the strongest compensatory effects on the ts phenotype and virus replication. Of these four lineages, lineage #6 contained six missense mutations, with none present at >45% of the reads, suggesting that this lineage contained mixed virus populations that might be difficult to study. Thus, this lineage was not evaluated further. Lineage #1 contained three prominent mutations, each present in >50% of the reads, in the NS1 ORF (I46M), the P gene-start signal (c2334a), and the M2-1 ORF (I87K). Lineage #3 contained eight prominent mutations, three of which were of >50% abundance: namely, one each in the P ORF (G26D), the M2-2 ORF (P4L), and the L gene-start signal (a8494g). Lineage #7 contained five prominent mutations, all with >90% abundance. Four of these were missense mutations in the NS1 ORF (S5T), the N ORF (M50L), the M2-1 ORF (E70D), and the L ORF (N1249K), and the other was a silent mutation in the L ORF.

Based on these observations, two derivatives of Min AL were made using mutations from lineage #1 (Fig 3A, top): one derivative contained the mutation M2-1[I87K] alone (virus Min AL-M2-1[I87K]) and a second derivative contained this mutation plus two additional mutations, namely NS1[I46M] and the P gene-start signal mutation (c2334a) (virus Min AL-M2[I87K]+2).

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Fig 3. Prominent mutations identified from the in vitro stress test incrementally reduce the temperature sensitivity of Min A.

A. Genome maps of Min AL and derivatives containing the reintroduction of one or more prominent (>50% abundance) mutations identified by whole-genome deep sequencing of passage 14 (P14) of lineages #1, 3 and 7 (Table 1). Reintroduced mutations are identified underneath the genome of each Min AL derivative and are described in the Results. The two viruses at the top contain the indicated mutations from lineage #1; the next three viruses contain the indicated mutations from lineage #3, and the bottom two viruses contain the indicated mutations from lineage #7. B-C. Multi-cycle replication kinetics of Min AL and derivatives. Replicate Vero monolayers in six-well plates were infected using an of MOI of 0.01 pfu/cell with wt RSV, Min AL, P14 supernatant from lineages #1, 3 or 7 of the in vitro stress test, or Min AL derivatives containing the indicated reintroduced mutations, and incubated at 32°C (B) or 37°C (C). Titers for Min AL, its derivatives, and wt RSV are the means with standard deviation of two replicate titrations each of two replicate wells, harvested at each timepoint as described in the legend of Fig 1C. Due to limited sample availability, titers of the P14 lineages #1 and #3 are the means of two replicate titrations of one well at every other time point. Day 0 titers are the back titration of the inocula. Min AL derivatives containing mutations from lineages #1 and #7 (B, C; right and left panels) were evaluated in a first experiment, and Min AL derivatives containing mutations from lineage #3 (B, C; middle panels) were evaluated in a second independent experiment. Wt RSV and Min AL were included in both experiments.

https://doi.org/10.1371/journal.ppat.1012198.g003

Three additional derivatives of Min AL were made using mutations from lineage #3 (Fig 3A, middle): one derivative contained the mutation P[G26D] alone (virus Min AL-P[G26D]); a second derivative contained this mutation plus two additional mutations, namely M2-2[P4L] and the L gene-start signal mutation (a8494g) (virus Min AL-P[G26D]+2); and a third derivative contained these three mutations combined with the M2-1[N88T] mutation (Min AL-P[G26D]+3).

Two additional derivatives of Min AL were made using mutations from lineage #7 (Fig 3A, bottom): one derivative contained the mutation M2-1[E70D] alone (virus Min AL-M2-1[E70D]), and a second derivative contained this mutation combined with three additional mutations, namely NS1[S5T], N[M50L] and L[N1249K] (Min AL-M2-1[E70D]+3).

All seven Min AL-derived viruses were readily rescued by reverse genetics and Sanger sequencing confirmed that the working virus stocks were free of any adventitious mutations.

Mutations identified in P14 of the in vitro stress test increased multi-cycle Min AL replication in vitro

We evaluated the effects of the introduced P14 mutations on the multicycle replication of Min AL at 32°C and 37°C. Vero cells were infected with an MOI of 0.01 pfu/cell and virus replication was monitored for 10 to 12 days (Fig 3B and 3C). Due to the large number of viruses to evaluate, two separate multi-cycle replication experiments were done. Min AL-derived viruses containing mutations from lineage #1 or #7 as well as the corresponding P14 of lineage #1 and #7 were evaluated in the same experiment (Fig 3B and 3C, left and right panels), while Min AL-derived viruses containing mutations from lineage #3 as well as P14 of lineage #3 were evaluated in a separate experiment (Fig 3B and 3C, center panels). Wt RSV and Min AL were included in both experiments.

At 32°C (Fig 3B), wt RSV reached peak titers of 6.9–7.1 log₁₀ pfu/ml on days 11–12 pi. Replication of Min AL was delayed and was maximal on day 12 pi (6.2–6.4 log₁₀ pfu/ml). Min AL-M2-1[I87K] from lineage #1 and all three Min AL-derived viruses from lineage #3 replicated to higher titers than Min AL, achieving peak titers similar to or surpassing those of wt RSV (6.9 to 7.5 log₁₀ pfu/ml). Min AL-M2-1[I87K]+2 and viruses from lineage #7 replicated to similar titers as Min AL (6.2 log₁₀ pfu/ml).

At 37°C (Fig 3C), differences were much more pronounced. Wt RSV reached peak titers of 6.4 to 7.2 log₁₀ pfu/ml. In contrast, Min AL replicated poorly, reaching titers below 2.5 log₁₀ pfu/ml. The incremental reintroduction of the selected P14 mutations increased replication of Min AL incrementally in the case of lineages #3 and #7. The individual mutations M2-1[I87K], P[G26D], and M2-1[E70D] from lineage #1, #3 and #7, increased Min AL titers by 128-, 9-, and 54-fold, respectively. Reintroduction of the additional set of mutations from each lineage recovered Min AL replication incrementally to P14 levels in the case of lineages #3 and #7. This showed that these mutations indeed were responsible for the partial loss of the ts phenotype of these Min AL lineages.

To determine whether the introduced P14 mutations reduced the temperature sensitivity of Min AL, we evaluated their effect on the T_SH of Min AL in a plaque assay on Vero cells at temperatures ranging from 32°C to 40°C (Table 2). This showed that, indeed, the incremental reintroduction of the P14 mutations from lineages #1 and #7 increased the T_SH of Min AL from 36°C to 39°C, and the reintroduction of the P14 lineage #3 mutations increased the T_SH to 40°C, confirming the compensatory effect of the P14 mutations on the ts phenotype of Min AL.

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Table 2. Temperature sensitivity of Min AL and derivatives on Vero cells.

https://doi.org/10.1371/journal.ppat.1012198.t002

Single cycle virus replication experiments to analyze the effects of P14 mutations on viral RNA, protein synthesis, and viral yield by Min AL

We next evaluated the effect of the P14 mutations on the expression of cell-associated viral RNA and proteins, as well as single-cycle viral yield, by Min AL derivatives during single-cycle replication experiments. These experiments were performed at 37°C (Figs 4 and 5), the temperature that had been shown in Fig 1C and 3C to have substantial differential effects on the replication of Min AL and its derivatives. To do so, quadruplicate wells of Vero cells per time point were infected with an MOI of 3 pfu/cell with wt RSV, Min AL, or the Min AL-derived viruses and incubated at 37°C. Due to the low titer of Min AL M2-1[E70D]+3 stock (representing lineage #7), this virus was not included in these evaluations. At 24 and 48 hpi, one well was harvested for total cell-associated RNA in order to evaluate the accumulation of positive- and negative-sense viral RNA (Fig 4); at 48 hpi, one well was used to harvest cells and evaluate viral protein expression by flow cytometry (Fig 5A and 5B); at 48 hpi, one well was used to collect cell lysates and evaluate protein expression by Western blot analysis (Fig 5C); and at 24 and 48 hpi, one well was used to harvest cell suspensions that were vortexed, clarified, and evaluated by plaque assay to measure single-cycle viral yield (Fig 5D). The results are described in the next two sections.

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Fig 4. Prominent mutations identified from the in vitro stress test incrementally rescued RNA synthesis by Min AL.

Vero cell monolayers in six-well plates were inoculated with an MOI of 3 pfu/cell with the indicated viruses at 37°C and the total cell-associated RNA was harvested at 24 and 48 hpi. Positive-sense RSV RNAs (primarily mRNA, with a small content of antigenome) were quantified in triplicate by strand-specific RT-qPCR using tagged primers (Materials and Methods). Data for the wt and CPD ORFs are shown with solid and hatched bars, respectively. Note that the sequences of the CPD ORFs (N, P, L) differed from the wt ORFs and therefore necessitated the use of separate primers and probes. Thus, taqman results involving the CPD N, P and L ORFs of the Min AL-derived viruses could not be directly compared to those of wt RSV. The accumulation of negative-sense genomic RNA also was evaluated using a strand-specific RT-qPCR assay with tagged primers and a probe specific to the M2-1 ORF, which was the unchanged wt sequence in all viruses and permitted direct comparisons. Data were normalized to 18S ribosomal (r)RNA and expressed as log₁₀ fold increase over Min AL at the 24 hour pi time point.

https://doi.org/10.1371/journal.ppat.1012198.g004

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Fig 5. Prominent mutations identified from the in vitro stress test incrementally and partially rescued protein expression and virus production by Min AL.

A-C. Protein expression. Additional replicate Vero cell monolayers from the single-cycle infection experiment described in Fig 4 (MOI of 3 pfu/cell, 37°C) were harvested at 48 hpi (one well per virus per time point) for analysis of viral protein expression by flow cytometry (A and B) and Western blot (C). (A and B) show data from the experiment in Fig 4 combined with two additional independent repeat experiments. Cells were permeabilized, immunostained for RSV N, P, G, and F protein expression, and evaluated by flow cytometry to determine the percentage of cells expressing the N, P, G and F proteins (A) as well as the level of expression of N, P, G and F proteins (expressed as median fluorescence intensity; MFI) in the N⁺P⁺G⁺F⁺ cells (B). Each experiment is represented by a symbol and means and standard deviations are shown. (C) Cell lysates were prepared and analyzed by Western blotting to evaluate the expression of P and F with tubulin used as a loading control. Ladder, molecular weight marker. D. Virus production. Additional replicate Vero cell monolayers from the experiment described in Fig 4 were harvested by scraping at 24 and 48 hpi (one well per virus per time point), vortexed, clarified, and titered in duplicate by immunoplaque assay to determine virus titer.

https://doi.org/10.1371/journal.ppat.1012198.g005

Single cycle virus replication: P14 mutations increased the accumulation of positive-sense viral RNAs by Min AL to wt RSV levels

From the single-cycle replication experiment described above (using Vero cells infected with an MOI of 3 pfu/cell at 37°C), viral RNA was extracted from cell lysates at 24 and 48 hpi, and gene-specific RT-qPCR taqman assays were used to quantify the accumulation of positive-sense N, P, G, F, M2 and L RNA as well as negative-sense genomic RNA (Fig 4). Note that intracellular positive-sense RSV RNA at these time points generally contains approximately 95% mRNA and 5% antigenomic RNA, and thus this assay was primarily for viral transcription. Because the nt sequences of the CPD and wt ORFs of the N, P, and L gene differed due to the CPD, different taqman assays had to be used to quantify CPD RNAs (Fig 4, hatched bars) and wt RNAs (solid bars), thus preventing direct comparison between Min AL and its derivatives with wt RSV based on these three genes. However, direct comparison between all viruses was possible for the G, F and M2 genes because they were unchanged in all viruses, as well as for the genomic RNA, which was quantified based on the M2 sequence that also was unchanged in all viruses.

Cells infected with Min AL at 37°C had low levels of accumulation of the various positive-sense viral RNAs at 48 hpi, indicating that the Min AL polymerase complex was inefficient at this temperature. For the other viruses at 24 and 48 hpi, the level of accumulation of each RNA was normalized relative to the Min AL value at 24 hpi taken as 1.0 (Fig 4). This analysis showed that the incremental addition of P14 mutations from lineages #1, 3, or 7 to Min AL increased the expression of all of the evaluated CPD and wt RNAs, and the complete sets of major mutations from lineages #1 (Min AL-M2-1[I87K]+2) or #3 (Min AL-P[G26D]+3) had the strongest effects. Indeed, at 48 hpi, the levels of positive sense G, F and M2 RNA in cells infected with Min AL-M2-1[I87K]+2 (lineage #1) or Min AL-P[G26D]+3 (lineage #3) were between 24- and 800-fold increased compared to Min AL at 24 hpi, and were comparable to wt RSV. This indicated that the sets of major P14 mutations increased the accumulation of positive-sense mRNAs by Min AL derivatives to levels that approached or equaled those of wt RSV at 24 hpi and, especially, at 48 hpi. In case of lineage #7, we were only able to compare the effect of the M2-1[E70D] mutation to wt RSV (because of the low titer of the other Min AL derivative bearing lineage #7 mutations). Its addition to Min AL moderately increased the synthesis of the positive-sense RNAs, similarly to the increases observed after addition of M2-1[I87K], an M2-1 mutation identified in lineage #1.

We also evaluated the accumulation of cell-associated negative-sense genomic RNA, representative of RNA replication. Accumulation of genomic RNA was undetectable between 24 and 48 hpi in cells infected with Min AL. Similarly to the positive-sense RNA, the incremental addition of the selected P14 mutations from lineages #1, 3, and 7 into Min AL increased the accumulation of negative-sense genomic RNA, and the complete sets of major mutations from lineage #1 or #3 had the strongest effect (30-fold increase compared to Min AL). However, the levels of genomic RNA in cells infected with Min AL-derived viruses remained 8- to 10-fold lower than in wt RSV-infected cells.

Single-cycle virus replication: P14 mutations increased Min AL protein expression and viral yield

From the single-cycle virus replication experiments described above (using Vero cells infected with an MOI of 3 pfu/cell at 37°C), we also evaluated the effects on Min AL of the P14 mutations on cell-associated viral protein expression and single-cycle viral yield. Viral protein expression was evaluated at 48 hpi by flow cytometry (Fig 5A and 5B). Data in Fig 5A and 5B are derived from additional wells in the experiment described in Fig 4 as well as from two additional independent experiments (n = 3 total).

For flow cytometry analysis, the infected Vero cells were harvested at 48 hpi, then fixed and stained under permeabilizing conditions with a cocktail of fluorochrome-labeled monoclonal antibodies to evaluate the expression of N, P, G and F proteins. Analysis was done on live single N⁺P⁺F⁺G⁺ cells.

At 48 hpi, only 3% on average of the cells infected with Min AL were positive for all four proteins by flow cytometry (Fig 5A). Similar to the viral gene expression, the incremental addition of P14 mutations from each lineage into Min AL increased the frequency of cells expressing all four viral proteins. The addition of the full set of selected P14 mutations from lineage #1 or #3 had the strongest effect, resulting in 51% to 63% of the cells expressing all four proteins. This remained lower than wt RSV, for which 97% of cells expressed all four viral proteins.

We also determined the median level of expression of N, P, G and F protein in N⁺P⁺G⁺F⁺ cells at this 48-hpi time point (expressed as median fluorescence intensity; MFI) (Fig 5B). Since the levels of viral mRNAs in cells infected with Min AL-derived viruses bearing the complete set of lineage #1 or #3 mutations had been found to approach or equal the levels for wt RSV at 48 hpi (Fig 4), it was of particular interest to determine whether the levels of expression of the viral proteins similarly approached or equaled those of wt RSV. In general, at 48 hpi, the MFIs of all four viral proteins remained substantially lower in cells infected with Min AL or derivatives than in cells infected with wt RSV (note that in Fig 5B, the level of expression of Min AL is set at 1.0 and values for the Min AL derivatives and wt RSV are expressed as fold-increases). For example, the MFI of the RSV N protein was about 5-fold lower in Min AL-infected cells than in wt RSV-infected cells, and the addition of lineage #1, 3, or 7 P14 mutations to Min AL did not substantially increase the N MFI closer to wt RSV levels; similarly, the MFI of the RSV P protein was about 2-fold lower in cells infected with Min AL or its derivatives compared to cells infected with wt RSV. Thus, while a higher proportion of cells was infected by Min AL derivatives compared to Min AL, the level of expression of N and P in individual cells was similar among Min AL viruses, and lower than in wt RSV infected cells. This might be due to the inefficiency of protein translation from CPD mRNAs.

Interestingly, in cells infected with the Min AL M2-1[I87K]+2 virus, which included the c2334a mutation in the P gene start signal, P protein expression was further reduced to less than half of that of Min AL (Fig 5B, see Discussion). In the case of the G and F proteins, which were encoded by ORFs not modified by CPD, the incremental addition of mutations from each lineage into Min AL increased the protein expression in infected cells by about 2- to 2.5-fold for the viruses that contained all the mutations from lineage #1 or #3. However, G and F expression still remained lower compared to wt RSV infected cells that showed 5- to 7-fold higher G and F expression than Min AL-infected cells.

From the single-cycle replication experiment described above, additional wells were harvested at 48 hpi for Western blot analysis using monoclonal antibodies against the RSV F, P (Fig 5C) and G (S1 Fig). G was only detected in Min AL P[G26D]+3 and wt RSV-infected cells and was predominantly detected as a diffuse band between 100 and 150 kDa, corresponding to the full-length, fully-glycosylated form. A few less-abundant bands (30–70 kDa) were also detected in wt RSV infected cells. These might represent intermediates with incomplete O-glycosylation or truncated versions of the G protein that are mostly present on virions released from infected Vero cells [38]. The blots were quantified by scanning, and the observed levels of P, F and G proteins were consistent with the flow cytometry results of Fig 5A and 5B.

Single-cycle viral yields of Min AL and derivatives were also evaluated. The incremental addition of mutations from each lineage to Min AL increased virus replication, following a trend comparable to that of the accumulation of viral genomic RNA and protein expression. However, the viral titers of the Min AL derivatives were at least 10-fold less at both time points compared to wt RSV.

Min AL and derivatives are highly attenuated in hamsters

We next evaluated the replication, immunogenicity, and protective efficacy of Min AL and derivatives in hamsters (see Fig 6A for the timeline of the experiment). Due to low titers of virus stocks, three Min AL derivatives were not included for analysis: Min AL P[G26D]+2 (lineage #3), Min AL M2-1[E70D] (lineage #7), and Min AL M2-1[E70D]+3 (lineage #7). Groups of 16 golden Syrian hamsters were inoculated IN with 6.0 log₁₀ pfu of the indicated virus. Hamsters did not show any clinical signs or symptoms of respiratory illness after inoculation with wt RSV, Min AL or Min AL derivatives, as typical for RSV in the hamster model [39]. Virus replication in nasal turbinates (NT) and lungs was evaluated from eight hamsters per group on day 3 pi. Lungs appeared, as expected, normal without gross pathology.

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Fig 6. Replication of Min AL and derivatives following IN inoculation of hamsters.

A. Timeline of the hamster experiment. Groups of 16 five- to six-week old golden Syrian hamsters were inoculated IN with 6 log₁₀ pfu per hamster of the indicated virus. An additional group of eight animals were kept uninfected as controls. At 3 days post-infection (dpi), eight hamsters per group were euthanized and nasal turbinates (NT) and lungs were harvested and homogenized, and viral titers were determined by immunoplaque assay. Serum was collected at day -2 and 28 dpi for evaluation of the anti-RSV antibody response. At 32 dpi, all remaining hamsters including the group of eight non-immunized control hamsters were challenged with wt RSV. At 3 days post challenge (dpc), all hamsters were euthanized, bronchoalveolar lavages (BAL) were collected for evaluation of the mucosal antibody response in the lower airways and NT and lungs were harvested for evaluation of the replication of the challenge wt RSV virus. B. Replication of wt RSV, Min AL and derivatives in NT and lungs at 3 dpi from eight hamsters per group. Harvested tissues were homogenized, clarified, aliquoted, snap frozen in dry ice and stored at -80°C. Titers were determined by immunoplaque assay and expressed as pfu/g of tissue. The limit of detection is 50 pfu/g of tissue (dotted line). Viruses are grouped by lineage (Li), i.e., the number of the in vitro stress test lineage in which the specific mutations were identified. The median, min, and max values, 25^th and 75^th quartile, and individual values are shown. Statistical differences in comparison to wt RSV are indicated at the top of each graph, while differences between Min AL and its derivatives are indicated in brackets (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001, Kruskall Wallis test with Dunns post hoc test).

https://doi.org/10.1371/journal.ppat.1012198.g006

In the NT, wt RSV replicated efficiently to a geometric mean titer (GMT) of 4.7 log₁₀ pfu/g (Fig 6B, left panel). Replication of Min AL was reduced by about 10-fold compared to wt RSV. Interestingly, the restriction of replication of Min AL increased with the incremental addition of the lineage #1 mutations. Specifically, introduction of mutation M2-1[I87K] alone (virus Min AL-M2[I87K]) reduced Min AL titers by about 3-fold and the further addition of the NS1[I46M] and P gene start (c2334a) mutations (virus Min AL-M2[I87K]+2) reduced Min AL replication by about 70-fold (p<0.01 compared to Min AL). The introduction of the lineage #3 mutations (viruses Min AL-P[G26D] and Min AL-P[G26D]+3) did not affect replication of Min AL in NT.

In the lungs, wt RSV replicated to a GMT of 3.9 log₁₀ pfu/g, as typically observed (Fig 6B, right panel). No or baseline replication of Min AL, Min AL M2-1[I87K] (lineage #1) and Min AL P[G26D] (lineage #3) was detected, showing that Min AL and derivatives were highly attenuated in the lung (p<0.0001 compared to wt RSV). However, the inclusion of further mutations from lineage #1 (Min AL M2-1[I87K]+2) and #3 (Min AL P[G26D]+3) partly rescued Min AL replication, with virus replication detectable in the lungs of 3 of 8 hamsters and 5 of 8 hamsters, respectively.

We also evaluated the expression of 13 inflammation-related genes by RT-qPCR at day 3 pi in the lungs of seven of the eight hamsters that were inoculated with Min AL or wt RSV (S2 Fig). Data are expressed as fold-increase over the expression determined from two unimmunized hamsters. This showed that, although wt RSV replicated to approximately 3.9 log₁₀ pfu/g in the lungs on day 3 (Fig 5B), it induced only a moderate inflammatory response, as only the interferon-induced gene MX-2 and the pro-inflammatory cytokine IL-1β were increased in lung tissues (10- and 5-fold increase compared to unimmunized animals, respectively). Min AL did not replicate to detectable levels in the lungs, as shown in Fig 5B, and the level of expression of these two cytokines was lower in Min AL-inoculated animals (p<0.05 compared to wt RSV for the expression of MX-2; S2 Fig).

Min AL and derivatives induce robust serum anti-RSV antibodies

We next evaluated the immunogenicity of Min AL and derivatives (Figs 7 and 8). In the hamster experiment described above, sera were collected at day 27 pi from the eight remaining hamsters per group and the levels of IgG and IgA antibodies binding to the prefusion form of RSV F (pre F) or RSV G were determined by ELISA using recombinantly-expressed purified pre F and G proteins (Fig 7).

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Fig 7. Min AL and derivatives induced robust serum anti-RSV antibody responses in hamsters.

Sera from eight remaining hamsters per group including the group of non-immunized hamsters were collected at 28 dpi to evaluate the anti-RSV antibody response. A-B. Serum anti-RSV pre F (A) and G (B) IgG (left panels) and IgA (right panels) titers were determined by ELISA (IgG) or dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) ELISA (IgA). The limit of detection is two log₁₀. C. The neutralizing activity of the sera from the immunized hamsters was evaluated by determining the 60% plaque-reduction neutralizing antibody titers (PRNT₆₀), performed in the presence of complement. Viruses are grouped by lineage (Li), i.e., the number of the in vitro stress test lineage in which the specific mutations were identified. The median, min, and max values, 25^th and 75^th quartile, and individual values are shown. Statistical differences to wt RSV are indicated at the top of each graph, while differences between Min AL and its derivatives are indicated in brackets (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001, One-way ANOVA with Tukey post test).

https://doi.org/10.1371/journal.ppat.1012198.g007

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Fig 8. Min AL and derivatives induced robust mucosal antibody responses in the airways of hamsters and were protective against wt RSV challenge.

At 32 dpi, eight hamsters per group including the group of eight non-immunized control hamsters were challenged IN with 6 log₁₀ pfu of wt RSV. At day 3 pc, animals were euthanized and bronchoalveolar lavage (BAL), NT and lung tissues were collected from each animal. A-B. Anti-RSV pre F (A) and G (B) IgG (left panels) and IgA (right panels) titers in BAL were determined by ELISA (IgG) or DELFIA ELISA (IgA). The limit of detection is one log₁₀. C. Replication of wt RSV challenge virus was evaluated by plaque assay from NT or lung tissues harvested at day 3 pc. In each graph, the median, min, and max values, 25^th and 75^th quartile, and individual values are shown. Viruses are grouped by lineage (Li), i.e., the number of the in vitro stress test lineage in which the specific mutations were identified. Statistical differences to wt RSV are indicated at the top of each graph, while differences between Min AL and derivatives are indicated in brackets (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001, Kruskall Wallis test with Dunns post hoc test or One-way ANOVA with Tukey post test).

https://doi.org/10.1371/journal.ppat.1012198.g008

Surprisingly, despite the strong restriction of replication in hamsters, Min AL induced high titers of serum anti-pre F IgG and IgA that were comparable to those induced by wt RSV (Fig 7A). With the exception of the most attenuated Min AL-derived virus (Min AL M2-1[I87K]+2), which induced the lowest titers of anti-pre F IgG and IgA antibodies, the other Min AL derivatives induced high titers of serum anti-pre F IgG and IgA that also were comparable to those induced by wt RSV. Min AL and derivatives also induced robust levels of serum anti-RSV G IgG and IgA antibodies, however the titers induced by Min AL were significantly lower than those induced by wt RSV (p<0.01 and p<0.001 for IgG and IgA, respectively, Fig 7B). Among the Min AL derivatives, Min AL M2-1[I87K] and Min AL P[G26D]+3 induced higher titers of serum anti-RSV G IgG and IgA than Min AL, whereas the titers induced by Min AL P[G26D]+3 were not significantly different than those induced by wt RSV.

We also determined the serum 60%-plaque-reduction RSV-neutralizing antibody titers (PRNT₆₀) induced by the viruses (Fig 7C). Min AL and derivatives induced robust mean PRNT₆₀ ranging from 7.2 to 9.3 log₂ (for Min AL M2-1[I87K]+2 and Min AL P[G26D]+3, respectively) albeit these titers were significantly lower than those induced by wt RSV (10.8 log₂; p<0.05 to p<0.0001).

Min AL and derivatives induced robust mucosal anti-RSV antibodies and were fully protective against wt RSV challenge

In the hamster experiment described above, the remaining hamsters were challenged on day 32 with 6.0 log₁₀ pfu of wt RSV (Fig 6A). Similarly to our observations after immunization, hamsters did not show any clinical signs or symptoms of respiratory illness after challenge with wt RSV. At day 3 post challenge (pc), all animals were necropsied and bronchoalveolar lavages (BAL) were collected to evaluate the levels of anti-RSV pre F and G IgG and IgA binding antibodies in the lower airways (Fig 8A and 8B). As expected, lungs appeared normal without gross pathology. NT and lung tissues were collected, homogenized, and subjected to immunoplaque assay to determine the level of replication of challenge wt RSV (Fig 8C).

Remarkably, although the replication of Min AL and derivatives had been low or undetectable in the lungs of immunized hamsters, as described above, these viruses (with the exception of the most attenuated Min AL M2-1[I87K]+2 virus) induced robust titers of anti-RSV pre F IgG and IgA in the lungs that were not significantly different than to those induced by wt RSV (Fig 8A). Similarly to the serum, Min AL and derivatives induced detectable but lower levels of anti-G IgG and IgA than wt RSV (Fig 8B). As expected, no anti-RSV pre F and G IgG and IgA antibodies were detected in the lungs of control mock-immunized hamsters that were challenged with wt RSV (Fig 8A and 8B).

We next evaluated the protective efficacy of Min AL and derivatives against wt RSV challenge at day 3 pc (Fig 8C). Wt RSV replicated efficiently in the NT and lung tissues of hamsters from the mock-immunized group. However, with the exception of Min AL M2-1[I87K]+2, the most-attenuated virus, Min AL and the other derivatives were fully protective against detectable wt RSV challenge replication in NT and lung tissues. To confirm that Min AL-immunized hamsters were protected against inflammatory responses after challenge, we also evaluated the expression of 13 inflammation-related genes by RT-qPCR on day 3 pc. We selected lungs from 7 mock-immunized animals with robust RSV challenge virus replication, and 7 Min AL-immunized animals (no detectable challenge virus replication in this group). Data are expressed as fold-increase over the expression determined from two unimmunized unchallenged hamsters [40]. Similarly to the results from S2 Fig, only the interferon-inducible gene Mx-2 and the pro-inflammatory cytokine gene IL-1β were increased in mock-immunized animals on day 3 after RSV challenge (9- and 3-fold increase compared to unimmunized unchallenged animals, respectively; p<0.001 and p<0.01 for Mx-2 and IL-1β; S3 Fig), indicative of only a moderate inflammatory response in mock-immunized animals after RSV challenge. However, in Min AL-immunized animals, the level of expression of the 13 inflammation-related genes after wt RSV challenge was comparable to unimmunized unchallenged animals, indicating that Min AL was protective against RSV challenge, with no sign of inflammation after challenge.

Min AL-derived viruses are genetically and phenotypically stable in vitro

Finally, we evaluated the genetic stability and the stability of the temperature-sensitive phenotype of the four Min AL-derivatives that were evaluated in the hamster experiment described above by subjecting them to an in vitro stress test (Fig 9). Five 25-cm² flasks of Vero cells per virus were inoculated using an initial MOI of 0.1 pfu/cell. Three lineages per virus (in red) were passaged four times in parallel at a temperature that was one degree below the virus T_SH (indicated in Fig 9). The incubation temperature was increased by one°C (to the virus T_SH) and four additional passages were made, for a total of eight passages, corresponding to approximately two months of continuous culture. The other two control lineages (in black) were passaged 8 times in parallel at the permissive temperature of 32°C.

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Fig 9. Min AL derivatives were genetically stable during an in vitro stress test.

The stability of four Min AL-derived viruses (Min AL M2-1[I87K], Min AL M2-1[I87K]+2, Min AL P[G26D] and Min AL P[G26D]+3) was evaluated in an in vitro stress test. Five replicate cultures of Vero cells in 25 cm² flasks were inoculated using an initial MOI of 0.1 pfu/cell. Two replicate cultures (in black) were passaged eight times at the permissive temperature of 32°C. Three replicate cultures (in red) were incubated for four passages at one degree below the indicated virus T_SH, followed by four additional passages at the virus T_SH, representing two months of culture. Each lineage is represented by a different symbol. Flasks were harvested when extensive syncytia were observed or when the cells started to detach. One of five ml of clarified fluids from the previous passage was used to infect the following passage of fresh cells. Furthermore, aliquots of clarified virus from each lineage were snap frozen for virus titration by plaque assay at 32°C at the end of the experiment and sequencing. Whole genome Sanger sequencing was performed at the end of P8 for the lineages that were passaged at 32°C. Whole genome Sanger sequencing of two of three Min AL M2-1[I87K] lineages (upper left panel, red square and circle) was done at the end of P8 while sequencing of the third lineage (red triangle) was done at P7 instead of P8 due to low virus titer at P8. Due to low replication, whole genome sequencing of the other three stressed Min AL derived viruses could not be done, but the sharp drop in replication was indicative of stability.

https://doi.org/10.1371/journal.ppat.1012198.g009

All four Min AL-derived viruses replicated efficiently at the 32°C control temperature, reaching final titers at the end of P8 of between 4.7 and 7.4 log₁₀ pfu/ml (Fig 9). Whole-genome Sanger sequencing using viral RNA isolated from supernatants harvested at P8 from all lineages at 32°C showed that none of the Min AL derivatives accumulated prominent mutations.

For the lineages passaged at increasing temperature (Fig 9), the Min AL M2-1[I87K] lineages replicated efficiently at one degree below its T_SH (37°C), reaching 7.0–7.3 log₁₀ pfu/ml at P4 (upper left panel). The titers subsequently decreased during the four passages at the virus T_SH. Whole-genome Sanger sequencing of viral RNA isolated from supernatants harvested at P7 for one lineage and at P8 for the two other lineages did not reveal any prominent mutations. The three other Min AL-derivatives, (Min AL-M2-1[I87K]+2, Fig 9, upper right panel; Min AL P[G26D], lower left panel; and Min AL P[G26D]+3, lower right panel), exhibited strongly reduced replication when passaged at one degree below their respective T_SH and were completely restricted at their respective T_SH, indicating that they did not acquire mutations that rescued replication. Overall this experiment indicated that Min AL derivatives were genetically and phenotypically stable when serially passaged at the permissive temperature of 32°C and at their respective T_SH.

Ethics statement

The hamster experiment was approved by the National Institutes of Health Institutional Animal Care and Use Committee (IACUC).

Cell lines

African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies). Vero cells were maintained in 2% FBS during experiments. Baby hamster kidney cells that constitutively express the T7 polymerase (BSR T7/5) [43] were grown in GMEM media (Gibco-Life Technologies) with 10% FBS and 2% non-essential amino acids (Gibco-Life Technologies). Every other cell passage, 2% of gentamicin (Quality Biological) was added to the media to maintain selection for the T7-polymerase-expressing cells. Both cell lines were maintained at 37°C with 5% CO₂.

Viruses

Min A, Min L and Min FLC viruses were described previously [33]. The parent virus of the CPD RSVs is the recombinant virus RSV D46/6120. RSV D46/6120 was derived from the original recombinant version of wt RSV called D46, which has a 15,223-nucleotide genome (Genbank accession number KT992094). RSV D46/6120 has an identical sequence except that it contains a 112-nucleotide deletion in the 3’ non-translated region of the SH gene and five silent nucleotide point mutations in the last three codons and termination codon of the SH ORF. These mutations were previously shown to stabilize the RSV cDNA during propagation in E. coli without any effects on the replication in vitro and in mice [44]. The sequence numbering in the present study is based on KT992094.

Construction and rescue of Min AL and derivatives

The Min AL antigenomic cDNA was generated by replacing the wt L ORF in Min A by the CPD L ORF from Min L using BamHI and KasI restriction sites. Mutations of interest identified during serial passage of Min AL were introduced into the Min AL antigenomic cDNA using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s recommendations. Min AL and derivatives were recovered by reverse genetics as previously described [33]. A second passage of the rescued viruses was performed on Vero cells to generate working P2 virus stocks. The consensus sequence of these virus stocks was confirmed by Sanger or Illumina deep sequencing using overlapping reverse transcribed PCR amplicons generated using previously described primers [33,35].

Virus titration by immunoplaque assay

As previously described [33], duplicate wells of Vero cells in 24-well plates were infected with 10-fold serial dilutions of virus for two h at 32°C. After seven to 10 days incubation at 32°C, cells were fixed with 80% cold methanol and immunostained using a mixture of three RSV F-specific monoclonal antibodies (MAbs) [28], and further incubated with a polyclonal goat anti-mouse-IgG(H+L) antibody conjugated to horseradish peroxidase (KPL, 5220–0341) to vizualise the plaques. Virus titers are expressed in pfu/ml.

Multi-cycle growth kinetics

Two sets of subconfluent wells of Vero cells in six-well plates were inoculated each in duplicate with the indicated viruses using an MOI of 0.01 pfu/cell. One set of plates was incubated at 32°C for 12 days while the other set was incubated at 37°C for 10 days.Virus was harvested on the indicated days pi by scraping and harvesting the cells with the media. The cell and media suspensions were vortexed three times for 10 sec each and the supernatant was clarified by centrifugation at 1,200 rpm for 5 min at 4°C. Aliquots of viruses were snap frozen in dry ice and stored at -80°C before titration by plaque assay.

Characterization of plaque size

As previously described [35], Vero cell monolayers in six-well plates were inoculated with 250 pfu per well of virus and incubated at 32°C for seven days with an overlay containing 0.8% methylcellulose. On day 7, plates were fixed with cold 80% methanol and incubated with a cocktail of three anti-RSV F MAbs [28] in Odyssey Blocking Buffer in PBS (Li-Cor), washed, and incubated with an R-phycoerythrin goat anti-mouse IgG(H+L) secondary antibody (Thermofisher, P-852). Plaques were visualized using the Celigo imager (Nexcelcom Bioscience) and the plaque size (μm²) was analyzed using Celigo software. An average of 3451 (±1200) individual plaques were analyzed per virus.

Illumina whole-genome deep sequencing

Viral RNA was isolated from clarified supernatants collected at the end of passage 14 (stressed lineages) or 18 (control lineages) of the Min AL in vitro stress test using the QIAamp Viral RNA extraction kit (Qiagen). Then, the viral RNA was reverse transcribed using Superscript II Reverse Transcriptase (RT, ThermoFisher) and the cDNA was amplified by PCR using overlapping RSV-specific primers and a high-fidelity DNA polymerase (pfx DNA polymerase, Thermofisher) as described previously [36]. Overlapping PCR amplicons were purified using the QIAquick PCR Purification kit (Qiagen) and sequenced at the NIH Intramural Sequencing Center (NISC) using Illumina MiSeq. Only the sequences corresponding to the outer-most primers (nucleotides 1–23 and 15,174–15,223) were not directly determined.

Then, a reference sequence of the parental Min AL genome was prepared using the Bowtie 2 software [45]. Sequencing reads of Min AL and Min AL lineages were received as fastq files from NISC and were aligned to the Min AL reference sequence using Bowtie 2 with the very sensitive local parameter. Aligned reads were next converted from SAM to BAM files and then sorted based on genome position using SAMtools [46]. Sequencing reads were then sorted by their position on the flow cell and PCR duplicates were marked and removed using Picard Tools (https://www.broadinstitute.org/files/shared/mpg/plathumgen/plathumgen_fennell.pdf) with a maximum optical duplicate pixel distance of 2,500. After sorting reads and removing duplicates, the depth of coverage was measured with SAMtools (average coverage per lineage was 7,530 ±775 reads/nt) and single nucleotide variants were detected using LoFreq [47] with the default parameters. Analysis focused on prominent mutations, defined as detected in ≥30% of the sequencing reads.

Single-cycle infections

As previously described [35,36], replicate monolayers of Vero cells in six-well plates were infected using an MOI of 3 pfu/cell at 32°C or 37°C with the indicated viruses. Two h after inoculation, the monolayers were washed twice with PBS and replenished with fresh media. At 24 and 48 hpi, four wells per virus were harvested: (i) one well was processed for analysis of cell-associated viral RNA by strand-specific RT-qPCR, (ii) one well was harvested for analysis of viral protein expression by flow cytometry, (iii) one well was harvested for Western blot analysis, and (iv) the last well was harvested to quantify virus titer by plaque assay as described above for the multi-cycle replication experiments.

Strand-specific RT-qPCR

The cell-associated RNA from infected Vero cells from single-cycle infections was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s recommendations. Then, RNA was subjected to strand-specific RT-qPCR to quantify viral positive-sense (mRNA and antigenome) RNA and negative-sense genomic RNA, as described previously [33,35] with minor modifications. Briefly, five hundred ng of DNAse-treated RNA were reverse transcribed using SuperScript III First-Strand Synthesis System (Thermofisher) and a primer specific either to antigenomic/mRNA or genomic RNA. Each RT primer was linked to an unrelated oligonucleotide tag [36] to provide for specific amplification of the RT product at the following PCR step. Then, the cDNA was amplified by qPCR in triplicate with a primer corresponding to the oligonucleotide tag, a gene-specific reverse primer, and a probe. QPCR data were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA internal control that had also been subjected to RT-qPCR in parallel using random first-strand primers and a 18S rRNA Taqman assay (Thermofisher). Data was analyzed using the relative quantification software on the Thermofisher Connect platform and expressed as log₁₀ fold increase over the Min AL 24 h time point.

Flow cytometry

As described previously [35], Vero cells from single-cycle infections (MOI of 3 pfu/cell, 32°C or 37°C) were harvested at 48 hpi, stained using the Live/Dead Fixable Near-IR or Blue Dead Cell dye (Thermofisher), washed with PBS, and fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences). Then, cells were stained for 20 min at RT in Perm/Wash buffer (BD Biosciences) containing a cocktail of pre-titrated anti-RSV antibodies; namely an anti-RSV P monoclonal antibody (Abcam, ab94965) labeled in house with fluorescein isothiocyanate (FITC), an allophycocyanin (APC)-labeled anti-RSV N monoclonal antibody (Imgenex, NB100-64752APC), an anti-RSV G antibody (Abcam, ab94966) labeled with brilliant violet 421 (BV421, Biolegend), and a Biotin-labeled anti-RSV F monoclonal antibody (Millipore, MAB8262B-5). After incubation, cells were washed three times with Perm/Wash Buffer and incubated with a streptavidin-PE-labeled secondary antibody (BD pharmingen, 554061) for 20 min at room temperature. The cells were washed three times with Perm/Wash Buffer and resuspended in PBS. Data were acquired using a BD Symphony flow cytometer (BD Biosciences) and analyzed using FlowJo 10.8. The expression of the virus proteins N, P, G and F was analyzed on single live cells.

Western blot analysis

Infected Vero cells from single-cycle infections were harvested in 1X NuPage LDS sample buffer (Thermofisher) diluted in PBS containing protease inhibitor (Roche). Cell lysates were homogenized using a QIAshredder spin column (Qiagen) and protein concentrations were determined by BCA assay (Pierce BCA Protein Assay kit). Thirty μg of proteins was denatured in a final composition of 1X NuPAGE LDS Sample Buffer (ThermoFisher) and 1X NuPAGE Sample Reducing Agent (Invitrogen) by heating at 90°C for 10 min before being subjected to electrophoresis on NuPAGE 4–15% Mini-PROTEAN TGX Gels (Bio-Rad) with 1X TGS Running Buffer (Bio-Rad). Odyssey Protein Molecular Weight Marker (Li-Cor) was run in parallel. Proteins were transferred to PVDF membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and stained with a primary mouse anti-RSV F (abcam, ab43812), a mouse anti-RSV P (abcam, ab94965) or a mouse anti-RSV G (abcam, 94966) antibody. The rabbit anti-Tubulin (abcam, ab52866n) antibody was used as a loading control. The secondary antibodies used were goat anti-rabbit IgG IRDye 680RD (at 1:15,000, Li-Cor, 926–68071), and goat anti-mouse IgG IRDye 800CW (1:10,000, Li-Cor, 926–32210). Membranes were scanned using Odyssey software, version 3.0 (Li-Cor).

Hamster experiment

On day -2, serum was collected from 104 five-to-six-week old male golden Syrian hamsters (Mesocricetus auratus) (Envigo). At day 0, six groups of 16 hamsters were inoculated IN under isoflurane anesthesia with 6 log₁₀ pfu of wt RSV, Min AL, or the indicated Min AL-derivatives. A last group of eight hamsters were left uninfected as control.

At 3 days pi, which corresponds to the peak of replication of wt RSV in hamsters, eight hamsters from each inoculated group were euthanized by carbon dioxide inhalation. Nasal turbinates (NT) and lungs were harvested and homogenized separately in Leibovitz (L-15) medium, and the suspensions were clarified by low-speed centrifugation and aliquots were flash-frozen and stored at -80°. Virus titers were determined later in duplicate by immunoplaque assay on Vero cells at 32°C using a methylcellulose layer contaning 1% Amphotericin B, 0.1% Gentamicin, and 0.06 mg/mL clindamycin phosphate. The limit of virus detection was 50 pfu/g in both the NT and lungs.

At 28 dpi, sera were isolated from the blood of all remaining hamsters to evaluate the RSV-specific antibody response. All sera were heated at 56°C for 30 min to inactivate complement prior to serology assays. The 60% plaque reduction neutralizing antibody titer (PRNT₆₀) was evaluated using a complement-enhanced immunoplaque assay as described previously [33].

At 32 dpi, the remaining hamsters were challenged IN with 6 log₁₀ pfu of wt RSV. Three days after challenge, hamsters were euthanized by carbon dioxide inhalation and NT, bronchoalveolar lavages (BAL) and lung tissues were harvested. Wt RSV virus titers in NT and lung tissues were determined in duplicate by plaque assay on Vero cells incubated at 32°C as described above. BAL samples were heated at 56°C for 30 min to inactivate complement prior to serology assays.

Expression of inflammatory and antiviral genes in lungs of hamsters

One hundred μl of lung homogenate was mixed with 300 μl of TRIzol LS (Thermo Fisher) and total RNA was extracted using Phasemaker Tubes Complete System (Thermo Fisher) and PureLink RNA Mini Kit (Thermo Fisher). RNA from lung homogenates of two control hamsters (non-immunized and non-challenged) was derived from a previous study [40]. Then, 7 μl of the extracted RNA was reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). TaqMan assays (Thermo Fisher) specific for hamster’s (Mesocricetus auratus) inflammatory and antiviral genes [48–50] were performed using the TaqMan Fast Advanced Master Mix (Thermo Fisher) as previously described [40]. Hamster β-actin was used as a housekeeping gene. qPCR results were analyzed using the comparative threshold cycle (ΔΔC_T) method, normalized to β-actin, and expressed as fold changes over the average expression of the two uninfected, unchallenged hamsters.