| Rabies,caused by rabies virus(RABV),is a strong contagious zoonosis without efficient curative.Rabies caused 100%death once clinical symptoms present.At least59,000 human dead from rabies every year although some countries and regions have eliminated rabies.RABV is an unsegmented,negative-stranded RNA virus and genome contains five structural genes that encode five proteins.Glycoprotein(G)is the key protein that determines pathogenicity and immunity of RABV.Therefore,pathogenicity and immunity of RABV could change through the modification of G protein and this may provide foundations for the research of mechanism of pathogenicity,prevention and treatment of rabies.RABV HEP-Flury strain has been used as animal vaccine widely in China and Japan because it's a highly attenuated strain.Recombinant HEP-Flury expressing IFN-?,IL6,two copies of G or rearranging G gene significantly increases the immunity of HEP-Flury.RABV strain GD-SH-01,a highly pathogenic virus,was isolated from the brain of a rabid pig and the reverse genetic system of GD-SH-01 has been constructed in our lab.Pathogenicity of HEP-Flury changes after the replacement of G gene with one of GD-SH-01.To further illustrate the role of G protein in pathogenicity of GD-SH-01,replacement of GD-SH-01 G gene with one of HEP-Flury was conducted and the recombinant strain r GDSH-HEPG was rescued.The results showed that r GDSH-HEPG caused no death of adult mice through intramuscular infection while 16.7%death of adult mice through intracranial infection,which illustrates the pathogenicity of r GDSH-HEPG was highly decreased.The important role of GD-SH-01 G protein in pathogenicity was further confirmed.To find single amino acid site in G protein that determines the pathogenicity of GD-SH-01,the amino acid sequences of G protein forom several RABV strains were compared.Amino acid position 19 in G(G19),G96,G132,G194,G243,G255,G333 and G349 were selected for mutation and the full amino acid of GD-SH-01 G protein was replaced with HEP-Flury.Mutant strains r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243,r GDSH-G255,r GDSH-G333 and r GDSH-G349 were rescued.Characteristics in NA cells,pathogenicity and immunity of all mutant strains were investigated to find key amino acid sites that determine the pathogenicity of RABV and clarify the mechanism of attenuation.G protein is the only membrane protein on the surface of RABV virion and plays important role in the reproduction of RABV.The pathogenicity,immunicity and reproduction of RABV may change after a single amino acid mutation in G protein.Recombinant viruses r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243,r GDSH-G255 and r GDSH-G333 show lower reproduction rate,but r GDSH-G349 higher,compared with GD-SH-01 through the test of growth curves in NA cells.Mutant strains r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243 and r GDSH-G255 spread lower than GD-SH-01 in NA cells and this is accorded with their decreased reproduction.The first step of replication of RABV is binding and invasion to cells.r GDSH-G194 and r GDSH-G255 show stronger ability of both binding and invasion than GD-SH-01 in NA cells according to the test of genome using q PCR.The binding rate of r GDSH-G243 and the ability of invasion of r GDSH-G349increase compared with GD-SH-01 in NA cells.This indicates that mutations at position G194,G243,G255 and G349 affects binding or invasion of RABV.However,The increased binding of r GDSH-G194,r GDSH-G243 and r GDSH-G255 does not contribute to virus reproduction,while the increased invasion of r GDSH-G349 may promote virus reproduction in NA cells.The genome copies of r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194 or r GDSH-G255 was lower than one of GD-SH-01 in NA cells after infection and this is accorded with the results of reproduction and spread.More transcription of type?interferon in NA cells infected with r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194 or r GDSH-G243 was detected than with GD-SH-01 and this may be related to higher expression of G protein.G protein is the main protein that affects cells apoptosis caused by RABV.Apoptosis caused by r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243 or r GDSH-G255 in NA cells is significantly lower than those by GD-SH-01 at both 24h and48h after infection through the flow cytometry test.Therefore,an amino acid mutation in G protein can affect apoptosis in NA cells.MTT assay indicate that all the mutant strains show less influence on cell viability of NA cells after infection compared with GD-SH-01.This is in accord with apoptosis in NA cells caused by RABV.This may be related to the decreasing reproduction and spread.Previous study has demonstrated that GD-SH-01induces apoptosis in NA cells through endogenous apoptosis pathway.In this study,mutation in G protein of GD-SH-01 affects apoptosis in NA cells after infection through endogenous apoptosis pathway based on the results that NA cells infected with mutant strain r GDSH-G255 showed lower activity of Caspase-3 and Caspase-9 compared with those infected with GD-SH-01.The pathogenicity of mutant strain was investigated in adult mice through both intramuscular and intracranial injection.The survival rate of mice infected intramuscularly with r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243,r GDSH-G255,r GDSH-G333 or r GDSH-G349 was 66.7%,50.0%,83.3%,83.3%,66.7%,100%,100%and 100%,respectively.The survival rate of mice infected intracranially with r GDSH-G19,r GDSH-G96,r GDSH-G132,r GDSH-G194,r GDSH-G243,r GDSH-G255,r GDSH-G333 or r GDSH-G349 was 0%,0%,0%,0%,0%,100%,100%and 16.7%,respectively.The survival rates of mice infected with GD-SH-01 through both intramuscular and intracranial route are 0 while the survival rates of mice infected with HEP-Flury through both intramuscular and intracranial route are 100%.These results indicated that mutation of G19,G96,G132,G194 or G243 mildly decreases the pathogenicity.The mutation in G349 site significantly decreased the pathogenicity because r GDSH-G349 does not cause death of mice through intramuscular infection.In addition,16.7%mice survives post infection with r GDSH-G349 through intracranial route.The mutation of G255 or G333 significantly decreases the pathogenicity,the attenuation degree of pathogenicity is similar to HEP-Flury.The peripheral VNA in mice immunized with each mutant strain is more than 0.5 IU/m L and they are protected after challenged with lethal CVS-24.This indicates that attenuated strains from GD-SH-01 strain has the potential to be vaccine strains.rGDSH-G255 induces more CD19~+CD40~+B cells in lymph node than GD-SH-01through flow cytometry test.After intranasal infection,more CD3~+T cells infiltrats into the brain of mice infected with r GDSH-G255 than with GD-SH-01 through immunohistochemical test.In addition,more CCL3?INF-??IFN-?and Ig G?-L chain m RNA were determined in the CNS of mice infected intranasally with r GDSH-G255 than with GD-SH-01 through q PCR test.Therefore,the mutation of G255 attenuates the pathogenicity of RABV completely and induces stronger immune response.r GDSH-G255not only presents a better safety but also induces a protective immune response in the mice.To further investigate the genetic stability of mutation,r GDSH-G255 was passaged in NA cells and mice continuously.The mutation of G255 showes no reversion through sequencing.What's more,no mutation happens in other sequence of G protein.Therefore,the mutation of G255 in r GDSH-G255 is genetically stable.When the amino acid G255 of GD-SH-01 was mutated,the pathogenicity of GD-SH-01 significantly decreases.To further illustrate the role of G255 in pathogenicity,the amino acid G255 of HEP-Flury was replaced with corresponding amino acid of GD-SH-01.As the results shown,the pathogenicity of recombinant RABV does not significantly change.Peripheral VNA in mice immunized with r HEP-G255 was similar to those with HEP-Flury.In addition,VNA induced by r HEP-G255 can protect mice chanllenged with lethal CVS-24.Therefore,the mutation of G255 could attenuate pathogenicity of RABV while reversed mutation does not increased pathogenicity.This mutation could provide another excellent vaccine candidate virus.This study further confirmed the important role of G protein of GD-SH-01 in pathogenicity.We firstly confirmed the several amino acid sites in G protein affect bio-characteristics of RABV in NA cells.This study firstly indicated that the mutation of G255 or G349 significantly decreases pathogenicity of RABV.These results not only provide new sites for the research of pathogenicity of RABV but also safe,stable and efficient attenuated rabies vaccine candidates. |
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