| [Background] According to the influenza surveillance data of WHO,from the 45 th week of 2017 to the 13 th week of 2018,influenza B virus became the dominant strain of seasonal influenza globally,among which the Yamagata lineage dominated,which had a certain impact on the health of the population.For the prevention and treatment of influenza,Western medicine is immune prevention by injection of influenza vaccine and the use of chemical drugs to block the breeding process of the virus.Traditional Chinese medicine plays a role by inhibiting virus replication and regulating the body's inflammatory response.Chemical drugs are mainly based on neuraminidase inhibitors and M2 ion channel blockers.Traditional Chinese medicine mainly consists of single Chinese medicine and compound Chinese medicine.Reverse genetics plays an important role in the genetic study of influenza virus.The reverse genetics technology platform of influenza A virus has been widely constructed and applied,while the successful construction of the reverse genetics technology platform of influenza B virus has been rarely reported.[Objective] This research will investigate the genetic characteristics and epidemic causes of influenza B virus circulating in Guangzhou,Guangdong Province during 2017 and 2018,and explore the possible effect of the new mutations in the virus sequence on functional changes of the virus,hoping to provide scientific basis for influenza surveillance and prevention and drug treatment of influenza.In addition,a reverse genetics platform for influenza B virus was constructed to provide a basis for the research on the genes of influenza B virus and mechanism of traditional Chinese medicine against influenza B virus.[Methods] 1.Pharyngeal swab specimens from influenza patients at Guangzhou Sentinel Hospital from December 2017 to April 2018 were collected,and immunofluorescence method was used to detect influenza B virus samples,followed by virus isolation culture and viral RNA extraction.Hemagglutinin and neuraminidase,the genes of interest,were amplified by one-Step RT-PCR,and then the gene sequences were determined and analyzed.2.According to the results of sequence comparision analysis,9 strains of virus with unreported amino acid mutation site in neuraminidase genes were selected for in vitro drug sensitivity test of oseltamivir and peramivir to obserse the presence of drug-resistant virus strains.In addition,in vitro antiviral activity experiments were carried out with the solution of lianhuaqingwen capsule to observe the difference of inhibition of virus activity between oseltamivir,peramivir and lianhuaqingwen capsule.After infecting MDCK cells with the virus volume of 100TCID50,solution of oseltamivir,peramivir and lianhuaqingwen capsule were added with successive half-diluted concentration,and the cytopathic changes were observed and IC50 values were calculated after culturing.3.According to the results of sequence comparision analysis,9 strains of virus with unreported amino acid mutation site in hemagglutinin gene were selected for growth curve experiment to observe differences in proliferative capacity between virus strains.MDCK cells were infected with 0.01 MOI virus.And then the virus supernatants were recovered at 24 h,48h and 72 h.Respectively,the proliferative capacity of the virus strains were observed by plaque assay. 4.The p HW2000 vector was used as a backbone,eight recombinant plasmids containing each viral gene fragment were constructed,and the plasmids were mixed and transfected into 293 T and MDCK mixed cells.After culturing,the virus was passaged by infecting MDCK cells to complete the rescue of influenza B virus.[Results] 1.A total of 40 nasal/pharyngeal swab specimens of influenza B cases from 2017 to 2018 were isolated and amplified,and 38 strains were successfully isolated,of which 28 Yamagata strains and 10 Victoria strains were initially identified.The HA and NA genes of 38 influenza B viruses were sequenced and the phylogenetic tree was constructed for analysis.(1)HA evolutionary analysis showed that the HA genes of 28 strains of Yamagata influenza B virus all belonged to the branch of Yamagata Clade 3,and belonged to the same subbranch as the vaccine strain B/Phuket/3073/2013,and another subbranch was a similar strain B/Wisconsin/1/2010.All 10 strains of the Victoria influenza B virus belonged to the Victoria Clade 1A branch,among which there was no Guangzhou epidemic strain in the evolutionary sub-branch Clade 1A(?1,?2)represented by B/Hong Kong/286/2017 and B/Norway/2409/2017.(2)NA evolutionary analysis showed that the NA gene of 27 of the 28 strains of Yamagata influenza B virus belonged to branch of Yamagata Clade 3,and belonged to the same subbranch as vaccine strain B/Phuket/3073/2013.The 10 strains of Victoria influenza B virus belonged to the branch of Victoria Clade 1A.No Guangzhou epidemic strains were found in Clade 1A(?2)represented by B/Norway/2409/2017,while Guangzhou epidemic strains were found in Clade 1A(?1)represented by B/Hong Kong/286/2017.In addition,it was found the B/Guangdong/GZ20/2018 strain is a interline-reassortment virus of BY-HA/BV-NA.The amino acid sequences of HA and NA of 38 influenza B viruses were analyzed:(1)Amino acid sequence analysis of HA: 28 strains of Yamagata lineage virus(including B/Guangdong/GZ20/2018)had mutation in 11 amino acid sites on HA.All 28 strains carried L187 Q and M266 V mutation.In addition,mutation site of S93 P,R151K,N179 K,P342S,V421 A,N436S and T547 I were found.B/Guangdong/GZ14/2018 strain lacked amino acid 540.B/Guangdong/GZ40/2018 strain was found T136 I site mutation,located on the 120-loop epitope.10 strains of Victoria lineage virus had mutations in 7 amino acid sites on HA.All 10 strains carried I132 V,N144D and A214 T mutations,of which I132 V was located on 120-loop epitope and N144 D was located on 150-loop epitope.In addition,mutation site of A169 T,K227R,N248 D and P343 S were found.(2)Amino acid sequence analysis of NA: 27 strains of Yamagata lineage virus(not including B/Guangdong/GZ20/2018)had mutation in 22 amino acid sites on NA.26 strains(96.3%)carried I49 M mutations.19 strains(70.4%)carried R65 H mutations.24 strains(88.9%)carried I171 M mutations.25 strains(92.6%)carried D342 K mutations.The other two strains B/Guangdong/GZ16/2018 and B/Guangdong/GZ34/2018 carried D342 N mutations at amino acid 342.27(100%)strains carried K373 Q mutations,and 17(63%)strains carried S402 P mutations.In addition,mutation site of I6 T,F12L,V45 I,P48S,A67 V,G70E,P76 L,N144S,R186 I,F266L,E308 G,R315K,T372 N,T389I,K419 E,T437I were found.10 strains of Victoria lineage virus and the interline-reassortment strain B/Guangdong/GZ20/2018 had mutation in 12 amino acid sites on NA.All 11 strains carried mutations of I120 V,K220N,S295 R,N340D,and E358 K.8 strains(73%)carried D384 G mutation.3 strains(27%)carried V401 I mutation.In addition,mutation site of P48 T,T106I,I262 M,M369V,V422 I were found.2.Five of the nine selected strains showed varying degrees of drug resistance,and all five belonged to the Yamagata lineage.The inhibitory effect of lianhuaqingwen capsule on the in vitro activity of four strains with high degree of drug resistance was similar to that of the drug-sensitive reference strains.3.The Yamagata lineage strains B/Guangdong/GZ19/2018,B/Guangdong/GZ23/2018,B/Guangdong/GZ24/2018,B/Guangdong/GZ28/2018,and B/Guangdong/GZ29/2018 had stronger proliferative capacity than B/Guangdong/GZ40/2018.The proliferative capacity of Victoria lineage strain B/Guangdong/GZ39/2018 was more prominent than B/Guangdong/GZ11/2018.The proliferative capacity of the inter-line reassortant strain B/Guangdong/GZ20/2018 was weaker than other 8 strains.Overall,the Yamagata lineage strains were more powerful than the Victoria lineage strains in proliferative capacity.4.A reverse genetics platform for influenza B virus was successfully constructed.[Conclusion] The trivalent influenza vaccine strains recommended by WHO in 2017-2018 failed to have a good protective effect on the population in Guangzhou.Therefore,it is necessary to pay close attention to the variation of influenza B virus and the matching degree with the vaccine strain,so as to timely formulate a reasonable vaccine update and vaccination program,and carry out scientific and effective prevention and control of influenza.The phenomenon of drug resistance to neuraminidase inhibitors oseltamivir and peramivir in epidemic strains,and the unreported amino acid mutation sites between drug-resistant strains and drug-sensitive strains,while lianhuaqingwen capsule played the advantage of traditional Chinese medicine and had stable antiviral activity against drug-resistant virus strains.In addition,the Yamagata strain showed stronger proliferative capacity than the Victoria strain,and there were some differences in the proliferative capacity between strains of different lineages,and there were unreported amino acid mutation sites between strains of different lineages.The mechanism needs further verification. |
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