Attenuated Getah virus confers protection against multiple arthritogenic alphaviruses

Figures

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  Fig 1. GETV-3ΔS2, GETV-Δ6K and GETV-3ΔS2-CM1 are attenuated in vitro and are avirulent in suckling mice. (a) Schematic representation of the genomes of the constructed viable mutant viruses. Red triangles and solid lines point to the introduced mutations. (b) BHK-21 cells infected with WT GETV or its mutants were fixed at 12 hpi. GETV infection was visualized by IFA using an antibody against E1 of GETV. Images were acquired with a Nikon microscope; scale bar, 500 μM. (c, d) Multistep growth curves of WT GETV, GETV-CM1, GETV-Δ6K, GETV-3ΔS1, GETV-3ΔS2, and GETV-3ΔS2-CM1 on BHK-21 (c) and Vero (d) cells. Cells were infected at a MOI of 0.01. Due to the compact format, some error bars cannot be shown. P values indicating statistical significance of differences between titres of WT GETV and GETV-3ΔS2-CM1 measured in three independent experiments at 24 hpi are shown. (e, f) Plaque morphologies of the above listed viruses on BHK-21 (e) and Vero (f) cell monolayers. The cells were fixed and stained on Day 2 (BHK-21) or Day 3 (Vero) post infection. (g, h) Two-day-old mice (n = 10 per group) were subcutaneously inoculated with 3×105 TCID50 of WT GETV, GETV-CM1, GETV-Δ6K, GETV-3ΔS1, GETV-3ΔS2, or…

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  Fig 2. Immune response generated by infection with GETV-3ΔS2-CM1 protects mice against subsequent WT GETV challenge. (a) Schematic description of the experiment. Three-week-old ICR mice were infected with 1 × 105 TCID50 of WT GETV or GETV-3ΔS2-CM1. Antibody responses were measured at 14 and 28 days post infection. At 30 days post infection, the mice were subcutaneously challenged with 1 × 106 TCID50 of WT GETV. Serum samples and organs were collected at the indicated time points. (b) Viral titres in the sera of the WT GETV- and GETV-3ΔS2-CM1-infected mice (n = 5) at 1 and 2 days post infection were determined using a TCID50 assay. (c) GETV-specific IgG titres in serum at 14 and 28 days post infection measured by ELISA performed using recombinant GETV p62-E1 protein and presented as OD450 values. (d) Neutralizing antibody titres (PRNT50) in sera at 14 and 28 days post infection determined using WT GETV virions. (e) Viremia on Day 1 to Day 3 post-challenge. (f) Viral loads in lung, spleen, and muscle tissues on Day 3 post-challenge. (g) Specific splenocyte proliferation. Two weeks after immunization, mouse spleens (n = 3 per group) were collected, spleen cells were isolated and…

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  Fig 3. Immunization with GETV-3ΔS2-CM1 generates passive immunity that protects neonatal mice against WT GETV challenge. (a) Schematic description of the experiment. Six-week-old female ICR mice were immunized with 1 × 105 TCID50 of GETV-3ΔS2-CM1 or mock-immunized (n = 3 per group). At 28 days postimmunization, neutralizing antibody titres in the serum samples were measured by the PRNT assay (b), and then, mating was performed. Two-day-old mice from immunized and mock-immunized mothers (n = 10 per group) were challenged with 3 × 105 TCID50 of WT GETV, and survival (c) and weight changes (d) were monitored for 12 days. (e) Two-day-old mice from immunized and mock-immunized mothers (n = 10 per group) were challenged with 3 × 105 TCID50 of WT GETV. On Day 1 and Day 2 post-challenge, five mice per group were sacrificed, and viral RNA loads in the indicated organs were measured using RT-qPCR. For panel c statistical analysis was performed using the log-rank test. Panel a in this figure was created with BioRender.

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  Fig 4. GETV-3ΔS2-CM1 is attenuated in newborn piglets. One-day-old piglets not fed colostrum were infected with 5 × 107 TCID50 of WT GETV or GETV-3ΔS2-CM1 (n = 5 per group). Disease scores were monitored for 14 days; sera from surviving animals were collected on Day 14. (a) The disease scores are based on the presence or absence of diarrhoea, hind limb paralysis and depression. Piglets without signs of disease were scored as 0. (b) Body temperature and (c) survival of infected piglets. (d) Examples of the clinical manifestations of disease in piglets; pathological changes in the lungs and H&E staining (scale bar 200 μm) of lung tissues from WT GETV-infected piglets that reached the clinical endpoint are shown. GETV-3ΔS2-CM1-infected piglets were from an additional group; these animals were sacrificed on Day 3 post infection. (e) Viral RNA levels in sera from infected piglets. (f) Piglets (n = 3 per group) were infected with 5 × 107 TCID50 of WT GETV or GETV-3ΔS2-CM1 and euthanized on Days 2 and 3 post infection (WT GETV group) or on Day 3 post infection (GETV-3ΔS2-CM1 group). Viral RNA levels in different organs were measured using RT-qPCR. (g) A group of 1-day-old piglets (n = 4) was…

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  Fig 5. GETV-3ΔS2-CM1 is safe and immunogenic in pregnant sows. (a) Schematic description of the experiment. Pregnant sows were intramuscularly immunized with 1 × 105 TCID50 of GETV-3ΔS2-CM1 (n = 3) or mock-immunized (n = 2). Serum samples, nasal swabs, and rectal swabs were collected at the indicated time points. (b) Body temperatures of sows within 1 week after immunization. (c-e). Viral RNA copy numbers in the serum (c), nasal swabs (d), and rectal swabs (e) at 1–5 days postimmunization. (f) GETV-specific IgG titres in the cord blood of newborn piglets from immunized and mock-immunized sows and (g) in sera from sows at 1–5 weeks postimmunization were measured by ELISA. (h) Neutralizing antibody titres in the sera from sows at Days 14 and 28 postimmunization were measured by PRNT. (i) Titres of GETV-specific IgG antibodies in the maternal milk at 1–10 days after natural parturition were measured by ELISA. For f, g, and i the GETV-specific IgG titres are presented as OD450 values. Panel a in this figure was created with BioRender.

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  Fig 6. Passive immunization with GETV-3ΔS2-CM1 protects newborn piglets against WT GETV challenge. (a) Schematic description of the experiment. Note that the sows are the same animals shown in Fig 5a. Piglets (n = 6 per group) from GETV-3ΔS2-CM1-immunized and mock-immunized sows were fed milk from their mothers for 1 day, after which they were challenged with 5 × 107 TCID50 of WT GETV. Body weights and disease scores were monitored daily, and samples were collected at the indicated time points. (b) GETV-specific IgG titres in the serum of 1-day-old piglets fed colostrum from immunized and mock-immunized sows were measured using ELISA. (c) Disease scores, (d) survival, (e) body temperatures, and (f) average daily weight gains measured for 14 days after challenge. Viral RNA copy numbers in the serum samples (g), nasal swabs (h), and rectal swabs (i) at Days 1–5 after challenge were measured using RT-qPCR. (j) GETV-specific IgG titres in serum samples from animals from the immunized sow group and surviving animals from the mock immunized sow group at 7, 14, and 21 days after challenge were measured using ELISA. (k) GETV-specific IgG titres in serum samples of piglets (n = 6) from the…

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  Fig 7. Immunization with GETV-3ΔS2-CM1 protects mice from infection with other arthritogenic alphaviruses. Three-week-old ICR mice (n = 25) were immunized using 1 × 105 TCID50 of GETV-3ΔS2-CM1, mice in the control group (n = 20) received PBS. On Day 14 postimmunization, sera from immunized animals were collected, and neutralization titres against GETV (all 25 mice) as well as these against RRV, SFV, CHIKV, ONNV, and BFV (5 mice per virus) were measured using the PRNT assay (a). Two days later, the mice were subcutaneously challenged as follows: mice for which neutralization titres against SFV were determined received 1 × 104 TCID50 of SFV6, mice for which neutralization titres against RRV were determined received 1 × 106 TCID50 of RRV T48, mice for which neutralization titres against ONNV were determined received 1 × 106 TCID50 of ONNV, and mice for which neutralization titres against BFV were determined received 1 × 106 TCID50 of BFV. For each challenge, control (mock-immunized) mice (n = 5 per group) received the same dose of virus. Survival (b), body weight (c), and viral titres in serum on Day 1, Day 2 and Day 3 (d) after challenge with SFV. Body weight (e) and viral titres in serum…

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  Fig 8. Viable chimaeras based on GETV-3ΔS2-CM1 and SFV are avirulent and protect mice against SFV6 challenge. (a) Schematic representation of the genomes of SFV6, GETV-3ΔS2-CM1, 3ΔS2-CM1/nsR/SFV/E, 3ΔS2/nsR/SFV/ORF2, and 3ΔS2-CM1/nsR/GE/SFV. (b) Multistep growth curves (MOI = 0.1) of 3ΔS2/nsR/SFV/ORF2 and 3ΔS2-CM1/nsR/GE/SFV in Vero cells. (c-f) Three-week-old ICR mice (n = 5 per group) were subcutaneously inoculated with 1 × 104 TCID50 of SFV6, 3ΔS2/nsR/SFV/ORF2, 3ΔS2-CM1/nsR/GE/SFV, or with PBS. Survival (c) and weight change (d) were monitored daily for 14 days. (e) Viral titres in sera at Days 1–3 post infection. For SFV6-infected mice, the last time point was not included, as in this experiment all SFV6-infected mice died on Day 3 post infection. (f) Neutralization titres against SFV6 in the sera of 3ΔS2/nsR/SFV/ORF2- and 3ΔS2-CM1/nsR/GE/SFV-immunized mice at 14 days post infection. (g, h) 3ΔS2/nsR/SFV/ORF2, 3ΔS2-CM1/nsR/GE/SFV, and mock-immunized mice from the previous experiment (n = 5 per group) were challenged with 1 × 104 TCID50 of SFV6 at 14 days postimmunization. The survival of the animals was monitored for 14 days after the challenge (g). Serum samples were collected at 1,…