Generation of single-round infectious rotavirus with a mutation in the intermediate capsid protein VP6

Abstract

Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.

Keywords: VP6; rotavirus; single-round infection; vaccine.

Fig 1

Generation of a VP6-knockout rotavirus. (A) Generation of MA104 cells stably expressing VP6. VP6 expression was confirmed by Western blotting. (B) Growth of rSA11-VP6-KO. MA104-VP6 cells were infected with viruses at an MOI of 0.001 and then harvested at 72 hours post-infection. (C) Expression of viral proteins by rSA11-VP6-KO. Cells were infected with the virus at an MOI of 1.0. At 8 hours post-infection, cells were fixed and examined in an immunofluorescence assay.

Fig 2

Generation of VP6-mutated rotaviruses. (A) Structure of VP6. The ribbon diagram of the VP6 protein is based on Protein Data Bank (PDB) accession no. 1QHD. Domain B and domain H are indicated in blue and red, respectively. The A′-A″, D′-D″, and αA-I loop structures are highlighted in green, blue, and gray, respectively. (B) Construction of the VP6 gene. The number of amino acid residues is shown above. (C) Growth of the rescued recombinant viruses. MA104-VP6 cells were infected with viruses at an MOI of 0.001 and then harvested at 72 hours post-infection. (D) Growth of the recombinant viruses in MA104 and MA104-VP6 cells. Cells were infected with viruses at an MOI of 0.01 and then harvested at 24 hours post-infection. (E and F) Growth kinetics of rSA11-169-174_Linker. Cells were infected with viruses at an MOI of 0.01. (G) Passage of the rSA11-169-174_Linker in MA104 cells. Cells were infected at an MOI of 1.0, cultured for 7 days, and then freeze–thawed. Then, 10% of the cell lysate was inoculated onto new MA104 cells. This was repeated five times. The lysates were then titrated. The limit of detection (LOD) was 200 FFU/mL. Viral titers below the detection limit are plotted as half of the detection limit (100 FFU/mL). (H and I) Expression of viral proteins by rSA11-169-174_Linker. For Western blotting, cells were infected with viruses at an MOI of 10 and then harvested at 8 hours post-infection. For the immunofluorescence assay, cells were infected with viruses at an MOI of 1.0 and then fixed at 8 hours post-infection.

Fig 3

Generation of single-round infectious rotaviruses expressing foreign genes. (A) Construction of the VP6 and NSP1 gene segments used to generate foreign gene-expressing rotaviruses. The number of nucleotides (nt) or amino acid residues (aa) is shown above. (B) Growth of the rescued recombinant rotavirus. MA104-VP6 cells were infected with viruses at an MOI of 0.001 and then harvested at 72 hours post-infection. (C) Fluorescence image of ZsG expression by rSA11-169–174_Linker-ZsG. Cells were infected with viruses at an MOI of 1.0. At 8 hours post-infection, cells were fixed and examined in an immunofluorescence assay. (D) Kinetics of NLuc expression by rSA11-169–174_Linker-NLuc. Cells were infected with viruses at an MOI of 0.01. The cells were harvested at the designated times. (E) NLuc-based neutralization test using serum from a mouse immunized with rSA11.

Fig 4

Generation of single-round infectious rotaviruses harboring the VP7 segment of human rotavirus. (A) Growth of the rescued recombinant rotavirus. MA104-VP6 cells were infected with viruses at an MOI of 0.001 and then harvested at 72 hours post-infection. (B) Neutralization test using single-round VP7 monoreassortant viruses; serum from a mouse immunized with rSA11 (simian G3 genotype) was used.

Fig 5

Viral challenge experiments using mouse models. (A) Schematic showing animal challenge experiments in newborn mice. Three groups of 4-day-old BALB/c mice (n = 8–9/group) were orally infected with 1 × 10⁶ FFU of the virus. (B) Changes in body weight. Statistical significance was analyzed using two-way ANOVA. (C) Diarrhea symptoms. The graph shows the percentage of mice (per group) that had diarrhea. Statistical significance was analyzed using two-way ANOVA. P values < 0.05 were considered statistically significant (*P < 0.05). ns = not significant. (D) Schematic showing the animal challenge experiments using adult mice. Two groups of 3-week-old BALB/c mice (n = 18–19/group) were orally infected with 1 × 10⁷ FFU of viruses. (E) Viral RNA copy number in intestinal samples. Intestinal samples were homogenized and subjected to RNA extraction and qRT-PCR. Statistical significance was analyzed using a t-test. The P value is shown. The limit of detection (LOD) was 20 copy/mg tissue. Viral titers below the detection limit are plotted as half of the detection limit (10 copy/mg tissue). (F) Titer of infectious virions in intestinal samples. The tissue homogenate was titrated in MA104-VP6 cells. The LOD was 100 FFU/tissue. Viral titers below the detection limit are plotted as half of the detection limit (50 FFU/tissue). Statistical significance was analyzed using the Mann–Whitney test. The P value is shown.

Fig 6

Immunization of mice with the single-round infectious rotavirus. (A) Schematic showing the immunization protocol. Three groups of 4-week-old BALB/c mice (n = 5–7/group) were orally immunized with 1 × 10⁷ FFU of viruses. (B and C) Detection of serum IgG and IgA against rotavirus. Statistical significance was analyzed using a t-test. P values < 0.05 were considered statistically significant. ns = not significant. (D) Neutralization titer in serum from immunized mice. The neutralization test was performed using rSA11-NLuc. The neutralizing titer is calculated as the maximum serum dilution yielding more than 50% neutralization. The limit of detection (LOD) was 1:200. Neutralization titers below the detection limit are plotted as half of the detection limit (1:100). Statistical significance was analyzed using the Mann–Whitney test. P values < 0.05 were considered statistically significant. ns = not significant.