Genetic Stability Of Sabin Strain As Virus Seed For Production Of Inactivated Poliomyelitis Vaccine And Deep Sequencing Method Of Sabin Strain Virus

Poliomyelitis is widely identified to be an acute viral infectious disease with highly infectious and extremely harmful caused by poliomyelitis virus (PV). There is no effective treatment for the disease and the only way to prevent people from being infected is vaccination. Oral poliomyelitis vaccine (OPV) was widely used due to its convenient and cheap, but the vaccine-associated paralytic poliomyelitis (VAPP) and vaccine-derived poliovirus (VDPV) cases caused by OPV use have increasingly received great attention. Poliomyelitis vaccine inactivated (IPV) can avoid VAPP and VDPV, to gradually replace OPV with IPV for avoiding the VAPP and VDPV, becomes the emphasized issue in the Polio Eradication and Endgame Strategic Plan. After the eradication of polio around the world, the conventional IPV production is generally carried out facilities with bio-containment requirement more stringent. WHO encourages developing the IPV made from Sabin strain (sIPV). The genetic stability of Sabin strain is extremely important in the research of sIPV. In this study, we detected the genetic stability through PV infectious titer, D antigen and PV complete genome Sanger sequencing.Furthermore, it’s necessary to distinct vaccine between passed and failed the monkeys neurovirulence test (MNVT) when monitoring the quality of vaccine in production, and mutant analysis by polymerase chain reaction (PCR) and restriction enzyme cleavage (MAPREC) can accurately measure the amount of a nucleotide site mutation, however, MAPREC can only monitor mutations at a few genomic loci and miss mutations at other sites that could adversely affect vaccine quality. With the development of the molecular genetic testing technology, the next generation sequencing (NGS) make it possible to sensitive detection and quantification of all mutations in the entire genome of Sabin strain. In this study, we discussed the preparation method of Sabin strains NGS template, include how to remove the interference of background nucleic acid and amplify the low concentration sample, how to purify and choose the sequencing platform.Objective 1. To analyze the genetic stability of Sabin strain as a virus seed(sub-master and working virus seeds)for production of inactivated poliomyelitis vaccine as well as vaccine virus SO+4, SO+5 and SO+6.2. A preliminary approach to establishing the NGS sample preparation of Sabin strains, include preprocessing, amplification, purification, screening and the sequencing platform.Methods 1. The sub-master and working seeds of strains Sabin I, Sabin II and PfizerⅢ as well as vaccine virus were determined for infectious titer and D antigen content, of which the complete genomes were sequenced.2. Compared the common NGS sample preprocessing method, amplification method and purification methods, then selected the best way of Sabin strain.Results 1.The titers of Sabin strains of various passages were 7.62-8.26 lgCCID50/ml, while the D antigen contents were 30-128 DU/ml. Compared with the complete genome sequences of Sabin I (AY184219.1), Sabin II (AY184220.1) and SabinIII(AY 184221. 1)and attenuated strain of original seed(SO)in GenBank, the sub-master seed(SO+2), working seed(SO+3), vaccine seed virus(SO+4), SO+5 and SO+6 of Sabin strain showed no base mutation.2. It’s necessary that DNase I for sample pretreatment, and the best way to PCR is the sequence independent single primer amplification.Conclusion 1.No change was observed in the complete genome sequence of Sabin strain as virus seed and the vaccine virus compared with that of original seed (SO), while the virulence was homogeneous, indicating high genetic stability.2. The template preparation method of Sabin strain NGS was preliminarily established.