Fig 1.
Construction of Min AL and characterization in vitro.
A. Genome map of the newly constructed Min AL virus compared to wt RSV and the previously-described Min A, Min L and Min FLC viruses [33]. CPD and wt ORFs are shown in black and grey, respectively. The number of silent nucleotide substitutions introduced by CPD in each construct is indicated to the right. B. Multi-cycle replication kinetics in Vero cells. Cell monolayers in six-well plates were infected using an MOI of 0.01 pfu/cell with the indicated viruses and incubated at 32°C (left panel) or 37°C (right panel). At 24 h intervals, cells from duplicate wells for each virus were scraped into the medium, vortexed to release cell-associated virus, the suspensions were clarified by low-speed centrifugation, and the media supernatants were harvested, aliquoted, and snap-frozen. Virus titers were determined later by immunoplaque assay at 32°C. Titers correspond to the mean of two replicate titrations of virus from each of two replicate wells at each timepoint. Day 0 titers correspond to the back-titration of the inocula. C. Plaque sizes. Vero cells in six-well plates were infected with 250 pfu/well of the indicated virus and incubated under methylcellulose at 32°C. At day seven pi, plates were fixed and stained with a mixture of three anti-RSV F MAbs and a PE-labeled secondary antibody. The plaque area (in μm2) was evaluated on an average of 3451 (±1200) plaques per virus (** = p≤0.01, **** = p≤0.0001, Wilcoxon rank test with continuity correction post hoc test).
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 (TSH, 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.
Table 1.
Mutations detected at a frequency of >30% in eight of the ten Min AL lineages at the end of passage 14 (2nd passage at 38°C) as well as in each of the two controls at passage 18 (32°C).
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.
Table 2.
Temperature sensitivity of Min AL and derivatives on Vero cells.
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.
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.
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, 25th and 75th 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).
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, 25th and 75th 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).
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, 25th and 75th 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).
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 TSH, followed by four additional passages at the virus TSH, 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.