HIV-1包膜基因变异的体外研究

采用亚型测定、核苷酸和氨基酸序列测定和同源性分析等方法,观察了HIV-1 ⅢB毒株在实验室长期传代过程中包膜基因变异的情况.研究的毒株包括:经过实验室8年多连续使用而获得的毒株、在MT4细胞长期连续传代而获得的每间隔10代的毒株样品及多次更换宿主细胞传代而获得的毒株.主要结果有:(1)各种毒株包膜基因变异均不显著,核苷酸序列同源性均大于92%,变异距离均小于7.5%,且随着传代数增加核苷酸趋于稳定,代间同源性由92%上升至99%,而变异距离由7.5%下降为0.6%.(2)在传代过程中HIV-1亚型保持稳定,各种毒株均为HIV-1 B3亚型.结果显示HIV在体外长期传代培养的过程中变异不大,遗传性状稳定,可能是体外生长的环境十分稳定,缺乏机体免疫学压力.结果也提示体外长期传代HIV毒株仍然适用于各种HIV的应用研究,用长期传代的方法发展HIV-减毒活疫苗的可能性不大.     

两个猪瘟病毒野毒株gp55基因抗原编码区序列的分析及比较

利用反转录－PCR方法扩增了吉林省猪瘟病毒（HCV）两个野毒株gp55基因的主要保护性抗原编码区,并将其克隆到pGEM－T载体中,然后用Sanger双脱氧法测定了其核苷酸序列,并推导了其氨基酸序列。将测定的这两个HCV野毒株的部分序列（350bp）与国内外已知的HCV序列进行比较,结果表明：这两个野毒株的核苷酸序列的同源性为94．9％,氨基酸序列同源性为97．4％,与1985～1992年意大利中部分离4个野毒株的同源性明显高于其它HCV毒株,核苷酸同源性分别为97．2％～98．3％和94.0％～949％,氨基酸同源性分别为98．3％～991％和97.4％～98.3％,而与我国的HCV标准强毒株即石门株的核苷酸同源性仅分别为83.1％和83.1％,氨基酸同源性仅分别为90.6％和91.4％。因此认为吉林省这两个野毒株与意大利中部的4个野毒株具有密切的关系,而与石门株很可能来源不同。     

猪瘟病毒糖蛋白E0基因的克隆及其表达研究*

用ＲＴ－ＰＣＲＡ法扩增分别获得了中国猪瘟病毒强毒石门株和免化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ,并克隆到ｐＧＥＭ Ｔ载体中测定其核着酸序列和推导出其对应氨基酸序列;结果表明这两个毒株间的Ｅ０基因核着酸序列同源性为９５．３％,氨基酸序列同源性为９４％,有１４个残基的差异;与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的免化弱毒Ｃ株相应序列进行比较,所测石门病毒核苷酸序列与上述各株对应序列的同源性分别为９８．０％、９７．１％、９２．７％、８６．８％和９５．４％;氨基酸序列同源性分别为９     

戊型肝炎病毒细胞分离株部分核苷酸序列分析

用抗原捕获／多聚酶链反应（ＡＣ／ＰＣＲ）对戊型肝炎病毒（ＨＥＶ）细胞分离株ＭＪ９０和Ｒ２５基因组的部分核苷酸序列进行扩增,获得了与ＨＥＶ缅甸株ＥＴ１·１相同的ｃＤＮＡ扩增带。该ｃＤＮＡ扩增带纯化后用双脱氧核苷酸ＤＮＡ链末端终止法测序,ＣＪ９０、Ｒ２５株的核苷酸和氨基酸序列与ＥＴ１·１克隆的同源性分别为９９．６％、１００％和９９％、９９％,从而证明ＭＪ９０和Ｒ２５毒株为ＨＥＶ．     

重组CHO细胞C_(28)株S基因序列的测定及分析

目的测定重组CHO细胞C28株S基因序列,研究其遗传稳定性,并与已全基因序列测定的乙型肝炎病毒的S基因序列进行比较分析,预测和揭示现有疫苗株对当前疾病流行株的防病效果。方法从C28株中选取第22代、24代2、5代2、7代、28代2、9代3、0代、31代3、2代3、3代和34代细胞,根据GenBank中C基因型adr亚型乙型肝炎病毒的全基因序列设计引物。采用酚-氯仿法抽提CHO细胞基因组DNA,用PCR法扩增各代次细胞的S基因,回收700 bp左右的目的片段,克隆至pMD18-T载体上进行序列测定。利用生物学软件MEGA4.1和BioEdit进行S基因序列同源性分析,绘制系统进化树,分析与其他HBV病毒株S基因的同源性。应用实验动物测定C28株生产的重组乙型肝炎疫苗的效价。结果 C28株十一个代次之间S基因序列核苷酸和氨基酸同源性均为100%;C28株十一个代次S基因与其他病毒株S基因比较,与C基因型乙型肝炎病毒同源性最高,与其他基因型乙型肝炎病毒的核苷酸同源性达91.4%～95.1%,氨基酸同源性达84.5%～93.3%。免疫NIH小鼠结果显示5批重组乙型肝炎疫苗的效价均符合标准。结论 C28株S基因在传代及保存过程中具有较高的稳定性,对当前疾病流行株有较好的防病效果。     

猪圆环病毒2型分离毒株全基因组的克隆及序列分析

根据GenBank中猪圆环病毒2型（PCV－2）基因序列,设计两对特异性引物,用PCR方法从接种疑似PMWS仔猪病料的细胞中分段扩增2个分离毒株全基因组。将扩增片段克隆入pGEM-T easy载体,筛选获得含有相应片段的阳性重组质粒。对质粒中的插入片段进行测序拼接,获得2株均为1768bp的全基因组序列。应用DNA star序列分析软件,对所测PCV－2序列与GenBank中的国内外PCV毒株进行同源性比较,并绘制系统发生树,结果发现：所测两毒株之间的核苷酸序列同源性极高,可达99．8％,亲缘关系密切;与PCV－2参考毒株的同源性介于95．3％－99．7％之间,其中与美国的一株PCV－2同源性最高,分别为99．5％和99．7％,亲缘关系很近;与国内两分离株同源性分别为96．4％和98．8％－98．9％;与PCV－1参考毒株同源性仅为77．1％－77．5％。     

狂犬病病毒aG株全基因组序列测定及遗传稳定性分析

目的探究狂犬病病毒(Rabies virus,RV)aG株全基因组序列特征及遗传稳定性。方法严格按疫苗生产工艺进行传代,提取主种子批、工作种子批及疫苗原液病毒RNA,通过RT-PCR技术扩增全基因组各片段基因,然后分别将其克隆到p GEM-T载体中,并进行序列测定;采用DNAStar软件包对aG株与Gen Bank中4aGV参考株(JN234411)以及18株基因1型RV参考株进行同源性分析。结果 aG株全基因组由11 925个核苷酸组成,共编码3 600个氨基酸。疫苗原液与主种子批全基因组核苷酸和氨基酸同源性均为100%,而工作种子批与主种子批的核苷酸与氨基酸同源性分别为99.97%和99.92%;aG株与4aGV参考株全基因组核苷酸与推导的氨基酸同源性均为99.9%,其与18株基因1型参考株核苷酸与氨基酸序列同源性分别为84.2%~97.6%和93.7%~98.3%;aG株传代病毒与4aGV参考株全基因组氨基酸序列高度保守,且各主要功能区未发生变异。结论狂犬病病毒aG株在实验室长期生产传代过程中,全基因组遗传特性稳定。     

我国部分地区H9亚型禽流感病毒血凝素基因序列比较与遗传发生关系分析

为从分子水平掌握我国H9亚型AIV的遗传变异情况和流行规律,本研究汇集近年来从我国12个省、市、自治区的发病鸡群中分离到的23株H9亚型禽流感病毒,通过RT-PCR方法和核苷酸序列测定获得了23个毒株的HA基因cDNA核苷酸序列。核苷酸和推导的氨基酸序列同源性比较结果表明,这些毒株HA基因的核苷酸序列同源性为94．1％～100％,氨基酸序列同源性为95．4％～100%;将这23个毒株和来自亚洲及世界其它地区的另外31株的HA基因cDNA序列同源性进行比较发现,分离自香港的HK170499株与日本的2个毒株关系较近;氨基酸序列分析发现,CKGS199、CKTJ196、CKTJ296、CKSH300和CKBJ197五个毒株各发生了一个潜在的糖基化位点的丢失。54株H9亚型AIVHA基因55bp～1152bp的氨基酸序列分析发现,裂解位点尽管有10种基序,但本研究中的23株和近年来从我国大陆和香港地区的分离的毒株则均为RSSR↓GLF;构成受体结合位点的191位氨基酸有一个规律,即所有中国大陆毒株与部分香港毒株都为N,其它毒株均为H,141aa～143aa处的糖基化位点有与191aa类似的规律,即：凡是191aa为N的毒株,该处均为NVS（CKBJ194除外）,凡是191aa为H的毒株,则该处均为NVT;遗传发生关系分析,中国大陆毒株处于欧亚谱系的第一支。本研究结果表明近年来我国鸡群中H9N2亚型禽流感病毒的感染流行可能有一个共同的来源,这为制定防治该亚型禽流感流行的有效对策提供了重要的科学依据。     

猪瘟病毒石门株与兔化弱毒株E0糖蛋白基因的克隆及序列分析

参考已发表的猪瘟病毒序列,设计并合成了一对引物,应用ＲＴＰＣＲ 技术,扩增了猪瘟兔化弱毒（Ｈｏｇ ｃｈｏｌｅｒａ ｖｉｒｕｓ ｌａｐｉｎｉｚｅｄ Ｃｈｉｎｅｓｅ ｓｔｒａｉｎ , ＨＣＬＶ） 和石门强毒株的Ｅ０ 糖蛋白基因,并将其克隆到ｐＧＥＭＴ 载体中,测定了其核苷酸序列,并推导了其氨基酸序列。结果表明我国这两株强弱不同毒株Ｅ０ 糖蛋白核苷酸序列同源性和推导的氨基酸序列同源性分别为９５－０ ％ 和９４－３ ％ ,有１３ 个氨基酸的差异,ＨＣＬＶ 比石门株多了一个潜在的Ｎ糖基化位点。将我国这两株病毒与国外已报导的ＨＣＶ 毒株Ｅ０ 基因序列进行了比较,发现石门株与日本的两株毒株ＡＬＤ 和ＧＰＥ－ 同源性较高,核苷酸序列同源性分别为９７－４ ％ 和９６－５ ％ ,氨基酸同源性分别为９７－４ ％ 和９６－０ ％ ,而与欧洲Ｂｒｅｓｃｉａ 株和Ａｌｆｏｒｔ 株同源性较低,核苷酸同源性分别为９２－２ ％ 和８６－５ ％ ,氨基酸同源性为９５－２ ％ 和９２－５ ％ , ＨＣＬＶ 与ＡＬＤ、ＧＰＥ－ 、Ｂｒｅｓｃｉａ、Ａｌｆｏｒｔ 株核苷酸同源性分别为９５－６ ％ 、９４－９ ％ 、９１－３ ％ 、８５－５ ％ …     

中国不同来源狂犬病病毒野毒株G基因主要功能区的序列分析

对我国不同来源的狂犬病病毒野毒株８２０２、ＢＲＶ、ＭＲＶ的Ｇ基因４０５￣１１４６位核苷酸序列,进行了逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）扩增、克隆和序列测定,并应用计算机对这３个毒株的测定序列和已发表的中国人源毒株（ＣＧＸ８９）的相应序列进行了分析比较。结果表明,这４个中国不同来源狂犬病病毒的Ｇ基因同源性较低,８２０２与ＢＲＶ的核苷酸同源性只有７９．５％,与ＭＲＶ的氨基酸同源性亦只有８２．２％;同     

Serial human passage of simian immunodeficiency virus by unsterile injections and the emergence of epidemic human immunodeficiency virus in Africa

There is compelling evidence that both human immunodeficiency virus (HIV) types emerged from two dissimilar simian immunodeficiency viruses (SIVs) in separate geographical regions of Africa. Each of the two HIVs has its own simian progenitor and specific genetic precursor, and all of the primates that carry these SIVs have been in close contact with humans for thousands of years without the emergence of epidemic HIV. To date no plausible mechanism has been identified to account for the sudden emergence in the mid-20th century of these epidemic HIVs. In this study we examine the conditions needed for SIV to complete the genetic transition from individual human SIV infections to epidemic HIV in humans. The genetic distance from SIV to HIV and the mutational activity needed to achieve this degree of adaptation to human hosts is placed within a mathematical model to estimate the probabilities of SIV completing this transition within a single SIV-infected human host. We found that the emergence of even one epidemic HIV strain, following a single human exposure to SIV, was very unlikely. And the probability of four or more such transitions (i.e. HIV-1 groups M, O and HIV-2 subtypes A and B) occurring in a brief period is vanishingly small. We conclude that SIV cannot become a zoonosis, but requires adaptive mutations to become HIV. Some modern event must have aided in the transition of SIV to HIV. Our research indicates that serial passage of partially adapted SIV between humans could produce the series of cumulative mutations sufficient for the emergence of epidemic HIV strains. We examined the rapid growth of unsterile injections in Africa beginning in the 1950s as a biologically plausible event capable of greatly increasing serial human passage of SIV and generating HIV by a series of multiple genetic transitions. We conclude that increased unsterile injecting in Africa during the period 1950-1970 provided the agent for SIV human infections to emerge as epidemic HIV in the modern era.     

In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.     

Sequence Variation in the Gpl20 region of SHIV-CN97001 during in vivo Passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances （divergence, diversity） were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif （GPGQ） and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.     

Sequence Variation in the Gp120 region of SHIV-CN97001 during in vivo Passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.     

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.     

Sequence variation in the gp120 region of SHIV-CN97001 during in vivo passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage. Foundation items: CIPRA (U19 AI051915); 973 (2005CB-522903).     

人鼻咽癌克隆株体外传代过程的群体变化

将人低分化鼻咽癌克隆株CNE-2 Z-5-2-B_7在体外连续传代培养,从群体的角度观察了不同代数的细胞形态定量、DNA含量和体外增殖能力的变化。结果:(1)各细胞形态参数、DNA含量出现异质性,为多种瘤细胞亚群所构成,而且随传代过程变化、消长。(2)41代以细胞面积大、核大、核浆比小、DNA含量高、异质性明显的大细胞群体占优势。其形态特征与鼻咽低分化鳞癌的大核型相似。(3)81代主要为胞、核面积小、核浆比大、DNA含量高的小细胞群体,其体外增殖能力较第1代明显为高,形态及生长特征与鼻咽未分化癌相似。提示随着肿瘤的演进,如不给予影响(治疗),瘤细胞有恶性发展的内在倾向。     

分子克隆化禽网状内皮组织增生症病毒传染性及其前病毒全基因组序列研究

利用脂质体转染技术,将含有SNV株禽网状内皮组织增生症病毒 （REV）前病毒全基因组cDNA克隆质粒转染鸡胚成纤维细胞（CEF）.用对REV的单克隆抗体和抗REV env-gp90的鼠血清作间接免疫荧光反应,在原始的转染细胞及随后传代的细胞中均显示病毒特异性抗原.而且,在连续传代细胞中的阳性率明显升高.用REV特异性引物对进一步传代后的细胞基因组作PCR,也检测出REV基因组.这些结果均表明所得到的分子克隆化病毒具有传染性,因而也进一步证明所用的质粒克隆包含有具感染性的全病毒基因组.对该全基因组cDNA克隆进行酶切所获得的数个亚克隆进行测序,并将序列进行拼接,完成了REV全基因组序列.REV的这个传染性克隆将有助于进一步研究REV的分子生物学特性.     

火鸡疱疹病毒在鸡胚成纤维细胞上有效代次的测定及其免疫原性恢复的研究

本试验证明,火鸡疱疹病毒(HVT)FC126株在鸡胚成纤维细胞(CEF)传55代的病毒,仍有预防马立克氏病(MD)的效力,至第70代效力明显下降。若将HVT CEF第35代病毒复归火鸡连续传4代,能明显恢复其免疫原性,用C细胞传代则有效代次可达到75代。因而,此方法能有效地解决免疫原性下降的种毒的更新问题。本试验还为该种毒和疫苗的有效代次的使用范围提供了可靠依据。