Attenuation Molecular Basis Of Rabies Virus Flury Strain And Development Of Improved Rabies Vaccines

Rabies is an enzootic viral disease widespread throughout the world and causes severe destruction to the central nervous system. The annual number of human deaths worldwide caused by rabies is estimated to be 55,000, mostly in rural areas of Africa and Asia. To date, no effective medical therapy has been established for overt rabies. Once neurologic symptoms of the disease develop, rabies is fatal to both animals and humans. Vaccination is the only effective way to control rabies. Therefore, we should take a deep insight into the pathogenic and attenuation mechanism of rabies virus (RV) and development of new types of rabies vaccines.I.Attenuation molecular basis of rabies virus Flury strainAttenuated Flury RV low-egg-passage (LEP) and high-egg-passage strains (HEP) were established through serial passage in chicken brain, chicken embryos, and culture cells using a Flury RV isolated from a girl who died of rabies. LEP kills adult mice after intracerebral inoculation, while HEP causes only mild symptoms in adult mice. The molecular mechanisms associated with RV virulence are not fully understood. Therefore, the LEP and HEP strains, showing a high homology of the complete nucleotide sequence, appear to be useful for exploring the pathogenic and attenuation mechanism of RV.Sequencing analyses revealed that the genomes of the LEP and HEP strains share 99.3% nucleotide sequence identity, and a deduced amino acid homology of 99.8%, 98.3%, 99.0%, 97.8%, and 99.6% between the individual N, P, M, G, and L proteins, respectively. The two strains have identical intergenic regions between N/P, P/M and M/G, and one substitution in G/L intergenic region. Sequences of the 3′and 5′terminal non-coding regions, which include the recognition and initiation site of the viral RNA polymerase, were completely conserved in the two viruses. A total of 27 amino acid substitutions were found between these two strains.To investigate the molecular mechanism for virulence and attenuation of Flury RV, two infectious viruses, rLEP and rHEP, were rescued from the genomes of LEP and HEP respectively. The growth curves of the rLEP and rHEP strains were similar to wildtype LEP and HEP strains respectively, in both neuronal NA and non-neuronal BHK-21 cells. The rLEP and wildtype LEP strains appeared to be strongly neurotrophic having similar index values of 0.84 and 0.78, respectively. As expected, both rHEP and wildtype HEP did not show any neurotrophic characteristics. Virulence of rLEP, rHEP, wild LEP and HEP in adult mice was determined by different inoculation routes, intracerebral (i.c.), intranasally (i.n.), or intramuscularly (i.m.). The rLEP strain killed adult mice by any inoculation route. The MLD50 of rLEP after i.c., i.n. and i.m. inoculation were 1 FFU, 474 FFU and 3.4×105 FFU, respectively, which were comparable to those of wildtype LEP. In contrast, all mice survived from i.c., i.n. or i.m. inoculation, without exhibiting any neurological symptom except some slight body weight loss in the first few days post inoculation. These results suggested that the rescued viruses had similar biological properties and pathogenicities as their corresponding parental wildtype virus in adult mouse.We generated a HEP mutant virus, rHEPG333R, in which the Gln at G333 was changed to Arg. The in vitro neurotropism index of rHEPG333R was significantly increased from 0 to 0.8, indicating that rHEPG333R had acquired the neurotropism property. Infection of adult mice confirmed that rHEPG333R was lethal. The MLD50 by i.c., i.n. and i.m. inoculation routines were 0.3 FFU, 887 FFU and 1.4×10~6 FFU, respectively. Theses results indicated that the mutation at G333 is sufficient to cause HEP to become highly pathogenic in adult mice.However, rLEPG333Q, in which the Arg at G333 was changed to Gln, lost its neurotropism property and had a neurotropism index value of 0 in cell culture. Infection by the i.m. route with the maximum dosage of 3×10~6 FFU did not kill adult mice, and all surviving mice did not show any signs of neural disease. However, when inoculated by i.c. and i.n. routes, rLEPG333Q remained highly lethal in adult mice. The MLD50 by i.c. and i.n. routes were 36 FFU and 2.1×10~5 FFU, respectively. These results suggested that substitution of Arg with Gln at G333 only eliminated the peripheral neuroinvasiveness of LEP by i.m. inoculation but not its lethal phenotype in adult mice by i.c. or i.n. inoculation. Further analysis revealed that the Gln (CAA) at G333 of rLEPG333Q completely reverted to Arg (CGA) in all mice after one i.c. inoculation. Passage of the virus in NA cells showed that the Gln (CAA) at G333 in rLEPG333Q was not stably maintained in NA cells in vitro, and partially mutated back to Arg (CGA) within five passages. These results indicated that rLEPG333Q could not stably maintain the GR333Q mutation in mouse neural tissue and NA cells. The virus reverted back to regain its neurotropism and highly pathogenic phenotype. In contrast, rHEP stably maintained Gln (CAG) at G333, in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages.To investigate if a specific property of the G gene itself or if the genome background of LEP is responsible for the reversion of the Gln mutation at G333 to Arg, we constructed two chimeric viruses. The rHEP-G(L)333Q virus was generated by replacing the ORF of the G gene of HEP with that of LEP in which the amino acid at G333 was mutated from Arg to Gln. The neurotropism index of rHEP-G(L)333Q was 0 and was the same as that of rHEP. All mice inoculated with 105 FFU of rHEP-G(L)333Q survived the infection and did not show any signs of neural disease. Another chimeric virus, rLEP-G(H), was generated by replacing the G gene ORF of LEP with that of HEP. The titers of rLEP-G(H) in both NA and BHK-21 cells were lower than that of rLEP and similar to those of rHEP. The in vitro neurotropism index of rLEP-G(H) was 0. All mice inoculated with 105 FFU of rLEP-G(H) died within 12 days post-infection. Genome analysis revealed that G333 of rLEP-G(H) had changed to Arg (CGG) from Gln (CAG) in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages. In comparison, the Gln (CAA) at G333 of rHEP-G(L)333Q was stably maintained when propagated in mice brain or NA cells. These results strongly suggest that mutation from Gln to Arg at G333 does not happen randomly. Certain viral element(s) in the LEP genome backbone other than the G gene might be responsible for the instability of Gln mutation at G333. The low fidelity of the RNA polymerase of negative-strand RNA viruses is the major reason for virus mutation. For this reason, we constructed another chimeric virus, rLEPG333Q-L(H), in which the ORF of L gene of rLEPG333Q was replaced with that of rHEP. Multi-step growth kinetics analysis showed that rLEPG333Q-L(H) replicated at a relatively lower rate, about 10-fold lower than that of the rLEPG333Q. All mice infected by the i.c. route with rLEPG333Q-L(H) survived and showed no signs of neural disease except slight loss of body weight. Genome analysis revealed the Gln (CAA) at G333 of rLEPG333Q-L(H) was stably maintained when propagated in mice brain or NA cells. These results indicated that the L protein is responsible for the stability of the Gln mutation at G333 of Flury RV.Moreover, we employed the annexin V binding assay to investigate if the attenuation of Flury RV changed its ability to induce apoptosis in neural cells. The ability of the different RVs to induce apoptosis in infected NA cells at 24 hours post infection was compared. The percentage of cells that were positively stained by the FITC-labeled annexin V in rLEP- or rHEPG333R-infected NA cells was about 2.9±0.7% and 2.8±0.6%, respectively. These values were similar to that of uninfected cells (2.6±0.6%). In contrast, rHEP, rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) induced significantly higher levels of apoptosis in NA cells, and the percentage of the cells that were positively stained by FITC-labeled annexin V were 2.1, 2.4, 2.1, and 2.1 times higher respectively than that of the uninfected cells. Further, we compared the expression level of viral proteins in infected cells. The rLEP virus expressed similar amounts of G and N proteins to rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) in NA cells. Meanwhile, there was no significant difference in the expression level of G or N protein between NA cells infected by rHEP and rHEPG333R. These results indicated that the higher levels of early apoptosis in NA cells induced by the recombinant viruses containing G333Q may not be associated with an increase in the expression of G protein.Two chimeric strains rHEP-G(L)333Q and rLEPG333Q-L(H) were highly attenuated and genetically stable. Immunization and challenge study results showed that rHEP-G(L)333Q or rLEPG333Q-L(H) induced significantly higher VNA responses and more effective protection than rLEP and rHEP, indicating two viruses would be able to serve as modified live vaccine candidates.II.Development of improved rabies vaccinesInactivated rabies vaccines have a distinct advantage in the biological safety, however can not be widely in developing countries especially in rural areas due to its high production cost and relatively short immunization persistent period. Improvement of vaccine antigen production efficiency will help to reduce production costs, while improve the immune efficiency and extend vaccination interval.In this study, we generated a recombinant RV strain rLEP-G carrying double glycoprotein genes in the genome background of LEP strain. Biological analysis showed that introduction of an identical G gene did not affect virus replication in vitro and pathogenicity in adult mice, however, leaded to higher production of G protein in infected cells. The inactivated vaccine prepared from rLEP-G strain grown in BHK-21 cells produced higher VNA titer than that of wtLEP by intramuscular inoculation both in mice and dogs. The strong increase in immunogenicity in both mice and dogs makes rLEP-G strain a superb candidate for an inactivated RV vaccine.