Rescue Of Measles Virus From CDNA And Design Of Measles Virus Live Attenuated Vaccine Candidates By Inhibiting Viral Messenger RNA Cap Methytrasferase

Measles caused by measles virus belong to national statutory B infectious diseases,is an acute infectious disease.Measles virus can cause severe clinical symptoms and even death in patients.it is a serious threat to public health.Hu-191 measles virus used for measles immunization in all of the province in china,it has played a significant role in protecting children from measles virus infection.Although the reported measles vaccination coverage rate in China rise constantly,measles outbreaks still occurred in china in recent years.Domestic studies show that the protectively of Hu-191 measles vaccine is declining and it caused some unwanted adverse effects.In fact,many vaccinated infants and children in China experienced side effects ranging from skin rash,itching,and swelling,to high fever.Base on Hu-191-measle vaccine strain(MeV-Hu191)widely used in china,we can generate more safety and efficiency measles vaccine candidates by using reverse genetics systems to engineered Hu191-measles virus genome with mutations,deletions,and insertions.Measles virus(MV)was isolated in 1954,is an enveloped virus that includes an nonsegmented,negative-sense RNA genome,is a member of the family Paramyxoviridae in the order Mononegavirales.Process of transcription and replication of MV is under the control of ribonucleoprotein(RNP).RNP consisted of the nucleocapsid(N)protein,the phospho-(P)protein and the large(L)protein.is the basic unit of infectivity of MV.Replication-competent RNPs is the essential part to rescue RNA virus,the plasmids that can express viral anti-genome RNA,viral N?P?L protein under the control of the T7 RNA polymerase promoter transfected to the cell was extensively used for the recovery of MV virus.Messenger RNA modification is the essential issue in NNS RNA virus gene expression and replication.During viral RNA synthesis,NNS RNA viruses produce capped,methylated,and polyadenylated mRNAs.Cap formation is essential for mRNA stability,efficient translation,it is now firmly established that mRNA capping and methylation in NNS RNA viruses evolves in a mechanism distinct to their hosts and gene expression.Recent evidence suggests that Large(L)polymerase protein of NNS RNA virus included MV is thought to produce the majority of enzymatic activities related to viral transcription and replication.the L protein contains six conserved region(CR)numbered I to VI.CR VI is responsible for mRNA cap methyltransferase(MTase)activitySequence alignments between CR VI of NNS RNA virus L proteins and known MTases suggest that the SAM-binding residues of MV L include G1788,G1790,G1792.Three recombinant MV clone were constructed with single point mutation G1788A,G1790A or G1792A in their SAM binding sites,and their sequences confirmed,respectively.Two mutant MV clones,named rMV-Hu191-G1788A and rMV-Hul91-G1792.We found that both rMV-Hu191-G1788A and rMV-Hu191-G1792A were significantly more attenuated compared to parental rMV-Hul91 in vitro,rMVs carrying mutations in the SAM binding site were genetically stable,formed significantly smaller viral plaques,and had delays in CPE and replication kinetics.Cotton rat was inoculated intranasally with 5×106 PFU of virus in a volume of 100?l to test the replication of rMV-Hul91,intranasally inoculated with 1.0×106 PFU of rMV-Hu191,rMV-Hu191-G1788A,and rMV-Hu191-G1792A respectively to detect the immunogenicity of rMV-Hu191.rMV-Hul91-G1792A had a greater degree of attenuation in cotton rats compared to rMV-Hul 91-G1788A.Both recombinant viruses triggered significantly higher neutralizing antibody compared to rMV-Hu191,and provided complete protection against MV challenge.In this study,we established a more efficient method to assemble a full-length cDNA clone of MV-Hu191 without using restriction endonucleases.This rescue system was highly efficient as we were able to recover many mutants in the CR VI of L protein including the two recombinant viruses with amino acid substitutions in the SAM binding site.This reverse genetics system can facilitate the rational design of safer,more efficient measles vaccine candidates.Given the fact that the MTase domain is highly conserved in L proteins of all NNS RNA viruses,Thus,this novel attenuation strategy can be employed for other NNS RNA viruses.