We first performed a sequence alignment of the CR Ⅳ (MTase region) of the L proteins among NNS RNA viruses (Fig. 1). An MTase catalytic site (K-D-K-E tetrad) is conserved in the CR Ⅳ of all L proteins. In the methylation reaction, the MTase must bind to the methyl donor, SAM, in order to achieve methylation. A SAM binding site (GxGxG…D motif) is conserved in the L proteins of NNS RNA viruses. However, the SAM binding site of the L protein of MuV, Human parainfluenza virus-2 (HPIV2) and Newcastle disease virus (NDV) contains a naturally occurring mutation. Specifically, the first amino acid residue in the SAM biding site is an alanine but not a glycine. Sequence alignment indicated that amino acids including A1814, G1816, G1818 and D1892 responsible for SAM binding in MuV L protein; and amino acids K1792, D1917, K1953 and E1990 correspond to the catalytic K-D-K-E tetrads of the MuV L protein. We performed an alanine scanning mutagenesis in the SAM binding site and K-D-K-E motif in MuV L protein and each of these amino acid residues was mutated to alanine. Since the first amino acid of the SAM binding site is naturally an alanine (A1814), we mutated this alanine to glycine to create a SAM binding site which is conserved in the L proteins of most NNS RNA viruses. Primers used for mutagenesis are listed in Supplementary Table S1. Each of these mutations was introduced into plasmid pYES-MuV-S79 encoding a full-length cDNA clone of MuV vaccine strain S79.
Figure 1. Sequence alignment in the conserved region VI (CR Ⅳ) of the L proteins of NNS RNA viruses. The sequence of CR Ⅳ in the L protein was selected and compared using the Clustal Omega software. The coordinates were based on the amino acids 1790-1990 of the L protein of MuV-S79. The predicted SAM binding sites (GxGxG…D) are shown in orange and methyltransferase catalytic sites (K-D-K-E) are shown in grey. Representative members in the Paramyxoviridae (MuV, mumps virus; MeV, Measles virus; HeV, Hendra virus; NDV, Newcastle disease virus; CDV, canine distemper virus; PPRV, peste des petits ruminants virus; SeV, Sendai virus; RPV, Rinderpest virus; HPIV, human parainfluenza virus-2), Pneumoviridae (PVM, pneumonia virus of mice; HMPV, human metapneumovirus; BRSV, bovine respiratory syncytial virus; HRSV, human respiratory syncytial virus), Rhabdoviridae (VSV, vesicular stomatitis virus) were selected for sequence alignment.
Using the reverse genetics system, all eight rMuVs were recovered, which were named rMuV-S79-K1792A, rMuV-S79-A1814G, rMuV-S79-G1816A, rMuV-S79-G1818A, rMuV-S79-D1892A, rMuV-S79-D1917A, rMuV-S79-K1953A and rMuV-S79-E1990A. MuV-induced CPE were observed after 2-4 days (Fig. 2A). Subsequently, MuV NP protein was detected in Vero cells infected by each recombinant virus using an immunofluorescence assay (IFA) and Western blot (Fig. 2B and Supplementary Fig. S1A). The parent strain obtained the top NP antigen protein expression at 72 h after transfection, while rMuVs were delayed by 1-3 days (Supplementary Fig. S1B). The top NP antigen protein expression of rMuV-S79-D1917A and rMuV-S79-E1990A are similar to the parent virus, while the top expression of other rMuVs is lower than the parent virus (Supplementary Fig. S1C). The sizes of virus-induced plaques differed between the rescued parental and mutant viruses (Supplementary Fig. S2). Each recombinant virus was plaque purified, as demonstrated in Fig. 3 and 4A, rMuV-S79-K1792A (0.64 ± 0.11 mm), rMuV-S79-G1816A (0.66 ± 0.10 mm), rMuV-S79-G1818A (0.52 ± 0.13 mm), rMuV-S79-D1892A (0.18 ± 0.09 mm) and rMuV-S79-D1917A (0.48 ± 0.10 mm) formed significantly smaller plaques in diameter compared to the parental rMuV-S79 (0.82 ± 0.16 mm). Interestingly, rMuV-S79-A1814G (1.23 ± 0.18 mm) formed bigger plaques than the parental virus. Recombinant rMuV-S79-K1953A (0.85 ± 0.12 mm) and rMuV-S79-E1990A (0.70 ± 0.13 mm) had similar plaque sizes with the parental rMuV-S79 strain. Finally, the genome of each recombinant virus was amplified by RT-PCR and sequenced (Supplementary Fig. S3). The results showed that each rMuV contained only the designed mutation.
Figure 2. Rescue recombinant MuV mutants using reverse genetics. BHK-SR-19-T7 cells were co-transfected with a plasmid encoding full-length MuV antigenome and supporting plasmids encoding N, P and L genes. At day 3 post-transfection, cells were harvested and co-cultured with Vero cells for 24 to 96 h. Cell culture supernatants were used for next passage in Vero cells. A Specific cytopathic effects of rescued mumps virus on Vero cells. Scale bar: 50 μm. B Indirect immunofluorescence analysis of MuV rescued by Vero cells at 48 h post infection, red is labeled as mumps N protein and blue is labeled as Vero cell nucleus. Scale bar: 50 μm.
Figure 3. Plaque morphology of rMuVs. A plaque assay was performed in Vero cells for each recombinant virus. After 7 days of incubation, plaques were fixed and stained with crystal violet.
Figure 4. Growth characteristics of rMuV mutants. A Comparison of plaque size of recombinant virus. Fifteen plaques of each recombinant virus were measured and the average plaque diameter was calculated. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. B Growth kinetics of rMuV mutants. Vero cells were infected by each recombinant virus at an MOI of 0.1, virus was harvested at the indicated time points. Virus titers of the rMuV mutants were determined by multiple step growth curve plaque assay in Vero cells. C Replication of rMuVs in IFN-/IFN + cell lines. Confluent Vero (v) and Hela cells (h) were infected with rMuVs at an MOI of 0.1. After 72 h post infection, virus titers were determined by plaque assay.
We evaluated the replication kinetics of these recombinant MuV mutants in Vero cells (Fig. 4B). The virus growth of most rMuV-S79 mutants was delayed by 1 day compared with the parental rMuV-S79. However, rMuV-S79-A1814G replicated earlier than the parental rMuV-S79 and reached a peak titer 6.75 ± 0.26 log10PFU/mL. The peak titers of rMuV-S79-K1792A and rMuV-S79-D1892A were 5.7 ± 0.59 log10PFU/mL and 6.0 ± 0.89 log10PFU/mL, respectively, which were lower than that of rMuV-S79 (7.6 ± 0.22 log10PFU/mL). The rMuV-S79-K1953A and rMuV-S79-E1990A, with a similar plaque size as rMuV-S79, had a lower peak titer (5.55 ± 0.43 log10PFU/mL and 6.32 ± 0.15 log10PFU/mL) than that of the parental strain. The peak titer of rMuV-S79-D1917A was 7.33 ± 0.06 log10PFU/mL, which was comparable to the parental strain at 72 h after infection.
Next, we determined whether defects in rMuVs replication are cell type specific. Briefly, Vero (IFN-) and Hela (IFN +) cells were infected with rMuVs and virus titers were determined in 72 h after infection by plaque assay. As shown in Fig. 4C, the ratio of viral titer between rMuV-S79 in Vero and Hela cells was 42:1. The ratio of viral titer between rMuV-S79-A1814G in Vero and Hela cells was 40:1 similar to the parental strain. The ratios of viral titer between rMuV-S79-G1816A, rMuV-S79-G1818A, rMuV-S79-D1917A, rMuV-S79-K1953A and rMuV-S79-E1990A in Vero and Hela cells were 184:1, 133:1, 204:1, 170:1 and 84:1 respectively, which higher than that of the parental strain. rMuV-S79-K1792A and rMuV-S79-D1892A cannot be detected > 100 PFU/mL viral titer in Hela cells. These data indicated that the replication kinetics of rMuVs in Hela cells was reduced and the reduction of rMuVs was more obvious than the parent strain.
The parental rMuV-S79 induced significant CPE in Vero cells at 72 h post-inoculation (hpi). All rMuV-S79 mutants except rMuV-S79-A1814G had significantly delayed CPE comparing with the rMuV-S79 (Fig. 5). Recombinant rMuV-S79-A1814G developed an earlier CPE, formed large syncytia at 24 hpi and reached a maximum CPE at 48 hpi. These data showed that rMuV-S79-A1814G had an increased syncytial formation whereas all other rMuV-S79 mutants had a significant attenuation in viral replication compared to the parental rMuV-S79.
We then tested the genetic stability of these recombinant viruses in cell culture. All rMuV-S79 mutants were passaged 10 times in Vero cells. At each passage, the MTase region in the L gene was amplified by RT-PCR and sequenced. The results showed that the L gene in each passage retained the desired mutation. At passage 10, the entire genome of each rMuV-S79 mutant was sequenced. All viruses retained the desired mutations and no other mutations were found in the viral genome as showed in Supplementary Fig. S3. The mumps vaccines are produced in primary chicken embryo fibroblast cell (CEF), so we tested the genetic stability of these rMuVs in CEF cells. rMuVs carrying mutations were repeatedly passaged 3 times in CEF cell and the entire genome of each recombinant virus was amplified by RT-PCR and sequenced. No mutations were found in the genome. These results indicated that these rMuV-S79 mutants were genetically stable in cell culture.
We next tested the replication ability of these rMuV mutants in vivo. Briefly, five-week-old, specific-pathogen-free (SPF) cotton rats were anesthetized with sevoflurane and inoculated intranasally with 1 × 106 PFU of each rMuV-S79 mutant (except rMuV-S79-A 1814G) or the parental rMuV-S79. No weight loss, death or obvious clinical signs of respiratory infection was observed in cotton rats. At day 4 post-inoculation, cotton rats were euthanized, lung, spleen and brain tissues were collected for virus titration and histology. No infectious MuV was detected in spleen and brain tissues for all groups. Viral titer in the lung tissues of rMuV-S79-G1818A-infected cotton rats was 2.97 ± 0.15 log10 PFU/g (P > 0.05), which is similar to rMuV-S79-infected control group (2.75 ± 0.12 log10 PFU/g). Viral titer in the rMuV-S79-G1816A (2.25 ± 0.06 log10 PFU/g, P < 0.001) and rMuV-S79-E1990A groups (2.48 ± 0.10log10 PFU/g, P < 0.05) were significantly lower than that in the rMuV-S79 group. However, no infectious virus was detected in the lungs of rMuV-S79-K1792A, rMuV-S79-D1892A and rMuV-S79-K1953A groups (Table 1). Also, no significant histologic lesion was detected in the lung and brain tissues for all groups (Supplementary Fig.S4A, S6A). To determine the viral antigen distribution in lung and brain tissues, IHC was performed using an antibody against the MuV N protein. As shown in Fig. 6, viral antigen-positive cells were detected inside the alveolar cells in lungs of rMuV-S79-infected cotton rats. Interestingly, the virus antigen-positive cells were detected in the bronchiolar cells of the rMuV-K1792A, rMuV-D1892A and rMuV-E1990A infected group, which had lower viral replication kinetics. Fewer antigen-positive cells were detected at the alveolar cells in lung tissues of other rMuVs infected groups. No viral antigen was detected in brain tissues from all groups as shown in Supplementary Fig. S5A. These results showed that rMuV mutants were more attenuated in replication in cotton rats compared to the rMuV-S79 virus strain.
Group Viral titer in lung Percentage of infection Viral titer (log10 PFU/g) rMuV-S79 80 2.75 ± 0.12 rMuV-S79-K1792A 0 ND rMuV-S79-G1816A 100 2.25 ± 0.06 rMuV-S79-G1818A 80 2.97 ± 0.15 rMuV-S79-D1892A 0 ND rMuV-S79-D1917A 80 3.04 ± 0.10 rMuV-S79-K1953A 0 ND rMuV-S79-E1990A 100 2.48 ± 0.10 DMEM 0 ND ND: not detected; Data each group were the mean of 5 cotton rats ± SD
Table 1. Infectivity of rMuV mutants in cotton rats.
Figure 6. Immunohistochemical (IHC) staining of lungs of cotton rats infected with rMuV mutants. Cotton rats were intranasally inoculated with 1.0 × 106 PFU of each rMuV mutant and were sacrificed at day 4 post-inoculation. The right lung from each cotton rat (N = 45) was fixed with 4% formaldehyde and embedded in paraffin, sectioned at 4 μm and stained with monoclonal antibody against mumps N protein (Abcam, ab9880, 1/500 dilution in PBS) to determine the distribution of viral antigen. Arrows, antigen-positive cells. Scale bar: 50 μm.
Next, the immunogenicity of rMuV-S79 mutants was determined in cotton rats. Five-week-old SPF cotton rats were anesthetized with sevoflurane and inoculated intranasally with 1 × 106 PFU of each rMuV-S79 mutant or parental rMuV-S79 and serum samples were collected weekly for determination of neutralizing antibody titer. At week 4 post-vaccination, each group was challenged with 1.0 × 107 PFU of a wild type MuV strain. At 4 days post-challenge (dpc), cotton rats were euthanized; and lung tissues were collected for virus titration and histologic examination.
We compared the dynamics of neutralizing antibody responses after vaccination with each rMuV mutant. Figure 7A shows cotton rats vaccinated with the parental vaccine strain rMuV-S79 induced neutralizing antibodies at week 2 post-vaccination, reached a peak titer at week 3 and started to decline at week 4. Antibody titers in rMuV-S79-K1792A, rMuV-S79-D1892A, rMuV-S79-G1818A and rMuV-S79-K1953A were not detectable at week 2, suggesting a significant delay in antibody responses for those mutants. Recombinant rMuV-S79-K1792A and rMuV-S79-D1892A had significantly lower antibody titers than the rMuV-S79 group over the entire time period. Although rMuV-S79-G1818A and rMuV-S79-K1953A had a significant delay in antibody responses (P < 0.05) but reached a comparable peak titer with the rMuV-S79 group. Recombinant rMuV-S79-E1990A induced similar antibody responses to rMuV-S79 (P > 0.05). Importantly, neutralizing antibody titers in the rMuV-S79-D1917A group triggered significantly higher antibody titers in all time points comparing with the parental rMuV-S79 group (P < 0.05). Thus, these data demonstrated that rMuV-S79 mutants had variable impacts on antibody responses. Their ability of triggering neutralizing antibodies can be ranked as following: rMuV-S79-D1917A > rMuV-S79, rMuV-S79-E1990A > rMuV-S79-G1816A, rMuV-S79-G1818A > rMuV-S79-K1953A > rMuV-S79-K1792A, rMuV-S79-D1892A.
Figure 7. Neutralizing antibody titers triggered by rMuVs in cotton rats. A Dynamics of neutralizing antibody titers triggered by each recombinant MuV in cotton rats. The serum of the cotton rats was collected at 2, 3, 4, 5, 7 and 9 weeks after immunization and antibody titers were determined using a plaque reduction neutralization assay. Data each group were the mean of 5 cotton rats ± SEM. B Comparison of the highest neutralizing antibody titers induced by each rMuVs in cotton rats. C Comparison of neutralizing antibodies at week 3. D Comparison of neutralizing antibodies at week 4. E Protective ability of rMuV-S79 mutants in cotton rats. *P < 0.05; **P < 0.01; ***P < 0.001.
The peak antibody titer of the rMuV-S79-D1917 group was higher than that of the parental strain, however, the rMuV-S79-K1792A and rMuV-S79-D1892A groups had lower peak antibody titers than the parental strain; and the other groups were similar to the rMuV-S79 group (Fig. 7B). Importantly, neutralizing antibody titer in the rMuV-S79-D1917A group produced antibodies earlier and higher than that of the parental vaccine group (P < 0.05). We also analyzed the peak antibody titer either at week 3 or 4 post-immunization. At week 3, rMuV-S79-K1792A, rMuV-S79-D1892A and rMuV-S79-K1953A produced significantly lower neutralizing antibodies than the parental strain whereas rMuV-S79-D1917 produced a higher neutralizing antibody titer (Fig. 7C). At week 4, rMuV-S79-D1892A and rMuV-S79-K1953A produced antibodies similar to the rMuV-S79, while rMuV-S79-D1917 and rMuV-S79-K1792A had the same trend as week 3 (Fig. 7D).
For the unvaccinated challenged control group, an average virus titer of 3.35 ± 0.26 log10 PFU/g was detected in the lungs (Fig. 7E). In contrast, no MuV titer was detected in the lungs from cotton rats vaccinated with each rMuV mutant and challenged with wild type MuV. These results showed that all rMuV-S79 mutants provided complete protection against MuV replication in the lungs. No significant lung and brain histology lesion was found in all groups, even in the unvaccinated cotton rats (Supplementary Fig. S4B, S6B). A few of inflammatory cell infiltrations were found in the lung of cotton rats immunized with rMuVs.
For lung tissues from unvaccinated controls challenged with wild type MuV, viral antigens were found inside the alveolar cells of lung tissues in all the cotton rats immunized with rMuVs (Fig. 8) and the viral antigens inside the alveolar cells in cotton rats vaccinated with rMuV-S79-D1917A were fewest compared to others. A small amount of inflammatory cell infiltration could be observed around the antigen-positive cells, which may be related to virus clearance. No viral antigen was detected in brain tissues from all groups as shown in Supplementary Fig. S5B.
Figure 8. Immunohistochemical (IHC) staining of lungs of cotton rats vaccinated with rMuVs followed by MuV challenge. Cotton rats were immunized intranasally with each rMuV mutant. At week 9 post-immunization, cotton rats were challenged with 1 × 107 PFU of a wild type MuV strain and were sacrificed at day 4 post-challenge. The right lung from each cotton rat (N = 45) was fixed with 4% formaldehyde and embedded in paraffin, sectioned at 4 μm and stained with monoclonal antibody against mumps N protein (Abcam, ab9880, 1/500 dilution in PBS) to determine the distribution of viral antigen. Scale bar: 50 μm.