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Objective@#Influenza H1N1 subtype vaccine candidate strains from a 2015—2016 year epidemic strain in China were prepared and identified by themethod of classical reassortment.@*Methods@#The influenza H1N1 epidemic strain and H3N2 high-yield reassortant parental strain (X-157) were mixed and inoculated into embryonated chicken eggs by the classical reassortmentmethod . The negative selection of mixed culture virus was carried out with the antiserum of H3 protein and the antiserum of X-157 strain. Real-time PCRmethod was used to test the HA and NA genes. Restriction enzyme digestionmethod was used to identify the internal genes. HA and NA genes of selected strains were sequenced. The strain which HA and NA genes possessed the same amino acid constitution with the wild type virus was selected and immunized to ferret. Two-way test was carried out.@*Results@#Five strains with expected HA and NA genes were selected by real-time PCR. Internal genes were identified, with 4 strains had 6+ 2 constitution, 1 strain had 5+ 3 constitution. Comparing with the wild type virus, HA and NA genes of the 5 strains had no mutation. HA titer of reassortant strains was above 1 024. HI titer of the selected NO.12 reassortment strain reached 5 120, and two-way test was passed. The yield of reassortant strain was 64 times that of the wild type strain.@*Conclusions@#A circulating influenza A (H1N1) strain of influenza A (2015—2016) was successfully prepared in China and laid the foundation for vaccine storage and disease prevention and control.
ABSTRACT
ObjectiveTo study the biological characterization and the genetic background of circulating CA16 strains in mainland of China for the purpose of CA16 vaccine development in the future.MethodsCA16 strains were isolated from throat swabs of patients with hand-foot-mouth disease and identified by neutralization assay and RT-PCR.The genotype of these isolates were determined by sequence alignment and phylogenetic analysis of VP1 gene.The proliferation dynamics and the plaque morphology were observed when propagated in Vero cells.The pathogenicity of these CA16 isolates was evaluated by challenging newborn mice.ResultsIn this study,six CA16 circulating isolates,BJ-1-6 were obtained.The RT-PCR products were 150 bp amplified with the general enterovirus primers and 210 bp with CA16 primers respectively,which cannot be amplified by EV71 primers.Additionally,these isolates were identified to display some obvious proliferation dynamics and plaque morphology when propagated for 96 h in Vero cells.The diameter of plaques were about 1.5 to 2 mm for BJ-1,BJ-2,BJ-4,BJ-6,4-5 mm for BJ-3 and 3 mm for BJ5,the plaques were regular except BJ-3.All the six isolates can be neutralized by the convalescent serum of patient infected with CA16.The virus titer of different isolates propagated for five passages in Vero cells was 7.0LgCCID50/ml.The sequence alignment of VP1 gene demonstrated that the genotypes of BJ-2,BJ-4,BJ5 were C1 and BJ-1,BJ-3,B J-6 were C,3 comparatively.The genetic distance of the VPI gene from theseisolates suggested that they were highly genetic identity with the homology of 90％ in nucleotide and 99％ in dedicated amino acid respectively.However,a distinctive difference in pathogenic ability in neonatal mice was found that the suckling mice challenged with BJ-3 & BJ-5 were paralyzed 4-5 d and dead 6-7d postchallenge,compared with the control group without any abnormality in the during of 14 d.ConclusionThe circulating CA16 isolates in China have different biological characteristics,different pathogenic ability and similar genetic backgrounds,which is helpful for the development of a CA16 vaccine in the future.
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Cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients is a rare occurrence. However, the emergence of transmissible strains has been reported between unrelated individuals. We analyzed the genetic relationship among P. aeruginosa isolates from Brazilian CF patients and transmissible clones which are worldwide spread. The data does not indicate the presence of closely related variant clones.
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The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.