人二倍体细胞及Vero细胞培养流感病毒的实验研究
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摘要
流行性感冒是一种古老的传染病，是由流感病毒引起急性呼吸道传染病。流感病毒属正粘病毒科，分甲(A)、乙(B)、丙(C)三型，可引起大流行、局部流行和散发。流感病毒抗原易变异，传播迅速，流行广泛，人群的特异性免疫状况不稳定，发病率高，老年人及患有各种慢性病或体弱者容易出现严重并发症，病死率高。流感一般临床表现为发热、头痛、肌痛、乏力、鼻塞、咽痛和咳嗽，流感还能加重潜在疾病或者引起继发细菌性肺炎或原发流感病毒性肺炎。当流感发生大流行时，可引起人群住院率、死亡率上升。世界卫生组织认为，减少流感危害的主要措施是疫苗接种。传统的流感疫苗是利用鸡胚进行生产的。而鸡胚这种传统的培养方式存在许多不足，不能够满足大批量生产疫苗的需要。细胞基质培养病毒应用于疫苗生产有很多优越性，因此只有寻找新的流感病毒培养基质才能满足流感疫苗生产技术发展的需要。同时流感也是国际上第一个实现全球监测的传染病，所以寻找新的细胞培养基质也是有效地分离和鉴定流感病毒的基础，有利于流感病毒的监测和临床快速诊断。
第一部分人二倍体细胞培养流感病毒的实验研究
本实验对流感病毒在几种人二倍体细胞上培养的可行性进行了初步研究。将四株不同型别的流感病毒(A／Kunming／1／2005 Va H3N2、A／Wisconsin／67／2005H3N2、A／New Caledonia／20／1999 H1N1和B／Malaysia／2506／2004)分别接种于KMB_(17)细胞、2BS细胞和MRC-5细胞上，在无血清的条件下于不同pH值和不同胰酶浓度的维持液中35℃培养72小时后收获病毒，测定血凝效价。实验中所用四株病毒在KMB17细胞上培养，H3N2亚型毒株(A／Kunming／1／2005 Va和A／Wisconsin／67／2005)血凝效价明显高于另外的H1N1亚型(A／New Caledonia／20／1999)和B型(B／Malaysia／2506／2004)毒株。在2BS细胞上培养，H3N2亚型毒株(A／Wisconsin／67／2005)检测不到血凝效价，而对H1N1亚型(A／New Caledonia／20／1999)和B型(B／Malaysia／2506／2004)毒株则有一定的敏感性。MRC-5细胞对本两株H3N2亚型的毒株(A／Kunming／1／2005 Va和A／Wisconsin／67／2005)敏感度低。该细胞对另外两株其它型别的流感病毒A／New Caledonia／20／1999 H1N1和B／Malaysia／2506／2004有一定的敏感度。综上所述，认为上述三种人二倍体细胞不是分离培养流感病毒的最佳细胞基质。若想作为常规的流感病毒分离培养用细胞，仍需大量的适应性研究和条件摸索。
第二部分流感病毒Vero细胞适应株的传代稳定性研究
本实验将流感病毒Vero适应株A／Kunming／1／2005 Va H3N2第25代病毒在已进行优化的培养条件下在Vero细胞上进行连续传代，监测其各代次血凝效价的变化，以验证其在Vero细胞中的传代稳定性。并对连续传代后病毒进行了PCR检测以及透射电镜检测，以验证适应株的基因及病毒颗粒的完整性。经实验证明，该株病毒已能够在Vero细胞中稳定传代并保持较高的血凝效价以及其基因和病毒颗粒的完整性。
Epidemic influenza is an epidemic disease which has a long history. It is causedby influenza virus. Influenza virus belong to orthomyxoviridae, and were devided intothree type as A type、B type and C type. It could cause pandemic occurrence、localflow and sporadic cases. The antigens of influenza virus are easy to change. Influenzavirus spread fastly and abroudly. The immunal condition of the human is unstable.And the incidence of this disease is very high. The old and the weak have high fidelityand mortality. Clinical manifestations of the flu are fever、headach、myalgia、fatigue、snuffle、angina and caugh. The flu can accentuation potential disease and can causesecondary bacterial pneumonia or essential viral pneumonia. During the pandemicperiod, there is an increase of the hospitalization and the mortality. WHO commendedthat the most effective method for influenza prevention is vaccination. Theconventional influenza vaccine are produced by chick embryo, but the deficiency ofthis method do exist, it can't satisfact the requirement of the vaccine production foranswer the pandemic. Culturing influenza virus by cells has many advantages. Hence,seeking for the new kind of cells for influenza virus culturing can satisfied the need ofthe manufacture of influenza vaccine. At the same time, the flu is a disease which ismonitored at global range, so seeking for the new kind of cells for influenza virusculturing is the cornerstone of all ttie research on influenza, it is helpful for thequickly clinical detection or diagnose.
Part 1 Preliminary study on culturing Influenza virus with diploid cell lines
Culturing four strain influenza viruses(A/Kunming/1/2005 Va H3N2、A/Wisconsin/67/2005 H3N2、A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004) on KMB_(17) cells、2BS cells and MRC-5 cells in mediumwithout serum, in different pH and typsin solution, at 35℃. After 72 hours, harvestthe virus and detect the HA titer. On KMB_(17) cells, the HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are higher than the other two (A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004). On 2BS ceils, the HA titer of he HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are undetectable(=0). And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are not very high. On MRC-5cells, the HA titer of A/Wisconsin/67/2005 H3N2 is zero. And the HA titer ofA/Kunming/1/2005 Va H3N2 is very low. And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are just not very high. Aboveall, those three human diploid cell lines are not the best choice to be used in influenzavirus culturing. It also need a deep reaserch.
Part 2 Studies on the stability of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells.
In this experiment, the HA titer of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells were detected during the continuous passage. It can keep hightiter. And the eight fragment of genome of the influenza virus were detected by PCR.The viron were observed by electron microscopy to verify the integrity of thevirosome. It was confirmed that influenza virus A/Kunming/1/2005 Va H3N2 cankeep its integrity of the virosome and the stability on Vero cells.
第一部分人二倍体细胞培养流感病毒的实验研究
本实验对流感病毒在几种人二倍体细胞上培养的可行性进行了初步研究。将四株不同型别的流感病毒(A／Kunming／1／2005 Va H3N2、A／Wisconsin／67／2005H3N2、A／New Caledonia／20／1999 H1N1和B／Malaysia／2506／2004)分别接种于KMB_(17)细胞、2BS细胞和MRC-5细胞上，在无血清的条件下于不同pH值和不同胰酶浓度的维持液中35℃培养72小时后收获病毒，测定血凝效价。实验中所用四株病毒在KMB17细胞上培养，H3N2亚型毒株(A／Kunming／1／2005 Va和A／Wisconsin／67／2005)血凝效价明显高于另外的H1N1亚型(A／New Caledonia／20／1999)和B型(B／Malaysia／2506／2004)毒株。在2BS细胞上培养，H3N2亚型毒株(A／Wisconsin／67／2005)检测不到血凝效价，而对H1N1亚型(A／New Caledonia／20／1999)和B型(B／Malaysia／2506／2004)毒株则有一定的敏感性。MRC-5细胞对本两株H3N2亚型的毒株(A／Kunming／1／2005 Va和A／Wisconsin／67／2005)敏感度低。该细胞对另外两株其它型别的流感病毒A／New Caledonia／20／1999 H1N1和B／Malaysia／2506／2004有一定的敏感度。综上所述，认为上述三种人二倍体细胞不是分离培养流感病毒的最佳细胞基质。若想作为常规的流感病毒分离培养用细胞，仍需大量的适应性研究和条件摸索。
第二部分流感病毒Vero细胞适应株的传代稳定性研究
本实验将流感病毒Vero适应株A／Kunming／1／2005 Va H3N2第25代病毒在已进行优化的培养条件下在Vero细胞上进行连续传代，监测其各代次血凝效价的变化，以验证其在Vero细胞中的传代稳定性。并对连续传代后病毒进行了PCR检测以及透射电镜检测，以验证适应株的基因及病毒颗粒的完整性。经实验证明，该株病毒已能够在Vero细胞中稳定传代并保持较高的血凝效价以及其基因和病毒颗粒的完整性。
Epidemic influenza is an epidemic disease which has a long history. It is causedby influenza virus. Influenza virus belong to orthomyxoviridae, and were devided intothree type as A type、B type and C type. It could cause pandemic occurrence、localflow and sporadic cases. The antigens of influenza virus are easy to change. Influenzavirus spread fastly and abroudly. The immunal condition of the human is unstable.And the incidence of this disease is very high. The old and the weak have high fidelityand mortality. Clinical manifestations of the flu are fever、headach、myalgia、fatigue、snuffle、angina and caugh. The flu can accentuation potential disease and can causesecondary bacterial pneumonia or essential viral pneumonia. During the pandemicperiod, there is an increase of the hospitalization and the mortality. WHO commendedthat the most effective method for influenza prevention is vaccination. Theconventional influenza vaccine are produced by chick embryo, but the deficiency ofthis method do exist, it can't satisfact the requirement of the vaccine production foranswer the pandemic. Culturing influenza virus by cells has many advantages. Hence,seeking for the new kind of cells for influenza virus culturing can satisfied the need ofthe manufacture of influenza vaccine. At the same time, the flu is a disease which ismonitored at global range, so seeking for the new kind of cells for influenza virusculturing is the cornerstone of all ttie research on influenza, it is helpful for thequickly clinical detection or diagnose.
Part 1 Preliminary study on culturing Influenza virus with diploid cell lines
Culturing four strain influenza viruses(A/Kunming/1/2005 Va H3N2、A/Wisconsin/67/2005 H3N2、A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004) on KMB_(17) cells、2BS cells and MRC-5 cells in mediumwithout serum, in different pH and typsin solution, at 35℃. After 72 hours, harvestthe virus and detect the HA titer. On KMB_(17) cells, the HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are higher than the other two (A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004). On 2BS ceils, the HA titer of he HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are undetectable(=0). And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are not very high. On MRC-5cells, the HA titer of A/Wisconsin/67/2005 H3N2 is zero. And the HA titer ofA/Kunming/1/2005 Va H3N2 is very low. And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are just not very high. Aboveall, those three human diploid cell lines are not the best choice to be used in influenzavirus culturing. It also need a deep reaserch.
Part 2 Studies on the stability of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells.
In this experiment, the HA titer of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells were detected during the continuous passage. It can keep hightiter. And the eight fragment of genome of the influenza virus were detected by PCR.The viron were observed by electron microscopy to verify the integrity of thevirosome. It was confirmed that influenza virus A/Kunming/1/2005 Va H3N2 cankeep its integrity of the virosome and the stability on Vero cells.
引文
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1、流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传10代，其血凝效价是稳定的。
2、RT-PCR结合PCR检测，证明了流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传代产物的基因完整性。
3、通过电镜检测初步证明了流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传代产物的病毒颗粒的完整性。
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[12] Stephenson I, Zambon MC, Rudin A, et al. Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers. J Virol. 2006, 80(10):4962-4970.
[13] 常海艳，陈建军，方芳，陈则．流感病毒灭活疫苗新型佐剂——壳聚糖增强免疫作用研究．中国生物制品学杂志。2004，17(6)：383-395
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1、流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传10代，其血凝效价是稳定的。
2、RT-PCR结合PCR检测，证明了流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传代产物的基因完整性。
3、通过电镜检测初步证明了流感病毒Vero细胞适应株A/Kunming/1/2005 Va H3N2在Vero细胞上连续传代产物的病毒颗粒的完整性。
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[7] 王斌．ISCOMATRIX~(TM)佐剂：细胞和体液免疫应答的有效诱导剂，国外医学预防诊断治疗用生物制品分册，2005，28(5)：204-207
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[10] Jorg H. Friz, Sylvia Brunner, Max L. Birnstiel, et al. The artificial antimicrobial peptide KLKLLLLLKLK induces predominantly a TH2-type immune response to co-injected antigens. Vaccine, 2004, 22 (25-26) :3274-3284.
[11] Bracci L, Canini I, Puzelli S, etc. Type I IFN is a powerful mucosal adjuvant for a selective intranasal vaccination against influenza virus in mice and affects antigen capture at mucosal level . Vaccine. 2005, 23(23):2994-3004
[12] Stephenson I, Zambon MC, Rudin A, et al. Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers. J Virol. 2006, 80(10):4962-4970.
[13] 常海艳，陈建军，方芳，陈则．流感病毒灭活疫苗新型佐剂——壳聚糖增强免疫作用研究．中国生物制品学杂志。2004，17(6)：383-395