Study The Stability Of The Cold-passaged Human Respiratory Syncytial Virus

The aim of this research is to analyze the toxicity change and the stability of HRSV A₂ strain after passeaged in the low temperature,do the basic research for the HRSV vaccine development.The major experiments include the continuous passage culture of HRSV in KMB₁₇ cell at 32℃,the heredity stability of HRSV passeaged in the low temperature,the stability of HRSV which stored in the different temperature and treated by physical conditions,and the syncytium of HRSV observed by nucleic acid fluorescence dyeing.The culture of HRSV passaged in KMB₁₇ at 32℃has to 35 generations,the multiplication time of HRSV is about 7-10 days,and the infectious titer is between 7.0-7.5 IgCCID₅₀/ml.This indicated that HRSV had adapted the KMB₁₇ cell gradually, which lay the good foundation for the change of cell matrix used to cell culture(diploid cell should be used to cell culture) and the screening of attenuate strain.The observation of heredity stability includes immunogenicity,a step growth curve and nucleic acid sequence examination.We observed a step growth curve of the 5th,19th, 28^th and 34th generations which passeaged in the low temperature,the results also showed that the adaption of HRSV cold-passaged strain,namely the lesion of higher generations under low temperature got more and more short at 32℃and the infectious titer also got higher and higher.Moreover,We carry on animal experimentation in the 21^st and 26^th generations which passeaged in the low temperature,to detecting their immunogenicity.The result showed that the poisonous degree of HRSV which passeaged in the low temperature is not insufficient,It still caused the target organ appeared obvious inflammation and interstitial pneumonia,the antigenicity is not strong,and the titre of antibody is 1:2—1:8.The last,our made nucleic acid sequencing of G-protein gene with different generations of HRSV passeaged in the low temperature,through the DNA match of the 15^th,20^th and 25th generations,not found the gene of G-protein occurrenced mutation.Observed the stability of HRSV which stored in the different temperature and treated by physical conditions.The result showed that HRSV is instability,HRSV was instable stored in 37℃and room temperature,the infectious titer of HRSV respectively decreased 1.0-2.0 lgCCID₅₀/ml and 0.2-0.8 lgCCID₅₀/ml everyday.Stored in 22℃,the infectious titer of HRSV decreased slowly,the change of the infectious titers＜0.6 lgCCID₅₀/ml after stored 5 weeks,could stored for short time.In order to guarantee the stability of the infectious titer,HRSV needed to stored in -20℃or lower temperature.The the infectious titer of HRSV decreases considerably after treated by freeze-thawed,it decreased 1.30 lgCCID₅₀/ml everytime.In addition,the infectious titer of the HRSV decreases fastest after the treatment of ultrasonication.It reduced to the half of the origial titer after the frist time,after the second time the virus turned to be little infection.Whereas the instability of HRSV,take two different protective agents into various generation viruses,and investigate the change in the infectious titer of HRSV.The results showed that protective agents had stabilization effect to the thermal instability and frozen-thawing instability of HRSV.The syncytium of HRSV in different cells observed by nucleic acid fluorescence dyeing.The multiplication time of HRSV is fastest in Hep-2 cell among these cells,could observ the syncytium after 24h;The multiplication time of HRSV in Vero cell is next;it in KMB₁₇ is the most slowly,and also the shape of lesions is different from the two former.The reason is about the sensitivity of HRSV to different cells and various surface receptor types in cell.