流感病毒在无动物细胞的细菌共生培养基里的培养研究

流行性感冒病毒流行时,在一些病人的上呼吸道里,常可以见到流感病毒和一些细菌(如甲型链球菌、葡萄球菌、流感杆菌等)共存,以往的实验也证明它们的关系比较密切。此试验发现:流感病毒在仅有甲型链球菌生长的培养基中有自我复制的现象,共生培养的病毒经过一系列的病毒学实验方法,证明确实是流感病毒。流感病毒在共生培养基里与它在鸡胚里的生长曲线基本相同,共生培养基里,只要细菌生长,流感病毒就能生长,在不同的温度(15℃、22℃、37℃)条件下,流感病毒和细菌的生长趋势出现密切的平行关系。甲型链球菌以外的其它细菌,如乙型链球菌、肠球菌、葡萄球菌、酵母菌、革兰氏阳性杆菌等菌株也能与流感病毒共生,只有个别的菌株不共生。共生培养的流感病毒能在较低的温度(22℃)环境下保持它的活性达2个月之久;在8℃的环境里,流感病毒也能共生繁殖,经过长期低温共生培养,其致病性减弱;流感病毒和其它两种病毒能在同一共生培养基里共生繁殖。实验研究中还简要讨论了共生的机理和实际应用等问题。     

影响麻疹病毒血凝素滴度的某些因素的探讨

自从Peries等报告麻疹病毒具有凝集猴血球的特性之后,近来有不少作者证实了这一研究。但麻疹病毒的血凝滴度较低,作血凝抑制试验时,一般均采用理化方法将血凝素加以浓缩。我们曾对麻疹病毒的血凝性质进行研究,可以不经浓缩而获得滴度较高的血凝素。关于应用麻疹病毒血凝抑制试验检查麻疹抗体的研究,已有报告。现将影响麻疹病毒血凝素滴度的某些因素报告如下:     

甲型链球菌SN-34和SN-35体外抑菌作用的研究

目的观察甲型链球菌SN-34和SN-35的抑菌效果。方法在固体培养基上用复层琼脂法观测甲型链球菌SN-34和SN-35对化脓性链球菌32309-2和金黄色葡萄球菌26001的抑菌环大小;在液体培养基中,甲型链球菌SN-34和SN-35分别与化脓性链球菌32309-2和金黄色葡萄球菌26001共同培养,然后观察一定间隔（2h）细菌的数量变化,并绘制生长曲线。结果化脓性链球菌32309-2的生长明显受到抑制。结论仅甲型链球菌SN-34具有抑菌作用。     

无血清细胞培养在发热伴血小板减少综合征布尼亚病毒生长中的应用

目的建立无血清培养基培养Vero细胞制备发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的工艺。方法分别采用含10%牛血清的MEM(10%MEM培养基)和无血清M2培养基(SF-M2培养基)在方瓶中培养Vero细胞制备SFTSV,比较无血清与含血清培养基培养Vero细胞制备SFTSV在病毒滴度及病毒繁殖曲线之间的差异。在生物反应器里用无血清培养的方式进行工艺放大,收获病毒原液并进行检定。结果无血清培养的Vero细胞能够满足SFTSV培养需求,与含血清细胞培养相比,单位细胞病毒产量没有降低,达到30~60个活病毒/细胞。可以实现在生物反应器的工艺放大,病毒高峰时病毒滴度均在7.0lg PFU/m L以上。结论无血清细胞培养可以应用于SFTSV的培养,有利于降低疫苗生产过程中的纯化难度,提高疫苗安全性。     

麻疹病毒融合蛋白(F)和血凝素(HA)在痘苗病毒中的表达

将麻疹病毒F和HA基因插入到痘苗病毒中,分别处于痘苗启动子P7.5与P11控制下,获得重组病毒vLmF和vCmH。用抗F多肽抗体和HA单抗进行ELISA检测,结果表明,两株重组病毒均能表达相应的麻疹蛋白。蛋白印迹显示重组病毒表达产物在分子大小,蛋白切割和糖化方面与麻疹病毒糖蛋白一致。两株重组病毒分别免疫家兔都能产生较高滴度的麻疹抗体,这些抗体具有中和作用和血溶抑制作用。此外,vCmH产生的抗体还具有血凝抑制作用。     

高效价腮腺炎病毒抗血清的制备及检定

目的制备高效价腮腺炎病毒抗血清,用于麻腮、麻腮风、麻腮风水痘联合疫苗的病毒滴定及鉴别试验等指标的检定。方法将腮腺炎病毒ME株接种于SPF鸡胚尿囊腔中,优化病毒制备工艺条件,收获高滴度病毒原液,制备免疫抗原;采用皮下多点注射法免疫SPF豚鼠,制备抗血清并对其进行中和效价、中和能力以及特异性干扰试验的检定。结果 ME株腮腺炎病毒以102倍稀释接种鸡胚尿囊腔,培养5 d经-20℃预冷1 h后,收获的尿囊液病毒滴度最高;以其免疫豚鼠所制备的抗血清平均中和效价达1∶3 276,高于鸡抗腮腺炎病毒血清;当豚鼠抗腮腺炎病毒血清稀释度为1∶320时,可完全中和1 000 CCID50/m L的腮腺炎病毒;豚鼠抗腮腺炎病毒血清对Vero细胞、RK-13细胞及2BS细胞的生长,均未见干扰及细胞毒性作用;豚鼠抗腮腺炎病毒血清对异种病毒(麻疹病毒、风疹病毒和水痘病毒)滴度的干扰试验显示,各病毒滴度其试验组与对照组的差值均0.50 lg CCID50/m L,表明豚鼠抗腮腺炎病毒血清对麻疹病毒、风疹病毒和水痘病毒的滴度均无干扰;豚鼠抗腮腺炎病毒血清及鸡抗腮腺炎病毒血清对麻腮风水痘联合疫苗各病毒滴度均无干扰,且两种抗血清之间差异无统计学意义(P0.05)。结论采用优化后的病毒制备工艺条件及免疫方法,可获得较高效价的抗腮腺炎病毒血清,经检定和验证,均符合含腮腺炎成分疫苗检定抗血清使用要求。     

鸡胚细胞的传代培养和麻疹病毒的增殖

为了建立原代鸡胚细胞的传代培养工艺,探究传代鸡胚细胞对麻疹病毒的敏感性和适应性,本研究将原代鸡胚细胞进行传代培养,分别采用原代鸡胚细胞和传代鸡胚细胞培养麻疹病毒沪-191(Shanghai-191,S-191)株毒种,并对病毒收获液进行滴度检测和基因序列测定。结果显示,原代鸡胚细胞可稳定传代培养至第10代,各代次细胞生长趋势相似;第5代鸡胚细胞染色体检查为正常染色体核型;第8代鸡胚细胞成瘤性检查未见成瘤;采用第3、5代鸡胚细胞制备的麻疹病毒滴度水平高于原代鸡胚细胞,但无显著性差异(n=3,P>0.05),编码病毒核蛋白(nucleoprotein,N)和血凝素蛋白(hemagglutinin,H)的基因序列与S-191株完全一致,未发生变异。本研究证实,原代鸡胚细胞可进行传代培养,各代次鸡胚细胞对麻疹病毒的敏感性不变,产毒水平无显著差异,可用于培养麻疹病毒。     

同时表达麻疹病毒L4株HA和F蛋白及人白细胞介素2(IL2)的重组痘苗病毒疫苗株的构建

构建了同时表达麻疹病毒ＬＡ株ＨＡ和Ｆ基因及人白细胞介素２（ＩＬ２）的重组痘苗病毒疫苗株ＲＶＪＭＬＨＡＦＫＩＬ２。Ｗｅｓｔｅｍｂｌｏｔ结果显示,ＨＡ、Ｆ和人ＩＬ２基因均在痘苗病毒中稳定有效表达,且产物与天然蛋白相近。ＨＡ分子有糖化的７９ｋＤ和非糖化的田ｋＤ两种形式;Ｆ分子以６０ｋＤ的前体Ｆ０、４３ｋＤ的Ｆ１和１８ｋＤ的Ｆ２三种形式存在。表达产物的血凝效价为１：８,血溶活性ＯＤ５４０的测定结果是Ｏ３７。该重组病毒免疫新西兰白兔及Ｃ５７小鼠,可以产生抗麻诊病毒ＨＡ和Ｆ蛋白的特异性ＥＬＩＳＡ（１：６４４～１６００）、血凝抑制（１：２５６～５１２）、血溶抑制（１：８０～１６０）和ＮＴ（１：６４０）抗体。接种兔及裸鼠后的毒力反应,明显低于只表达ＩＬ２的重组痘苗病毒疫苗株ＲＶＪ１２３,表现为兔皮肤红肿范围小,持续时间短,皮肤无坏死;裸鼠毒力的结果,表现出ＲＶＪＭＬＨＡＦＫＩＬ２病毒只存留于接种局部,而且滴度大大降低,未发现该病毒向其它脏器播散和增殖。麻疹重组痘苗病毒疫苗株的构建为该疫苗株的人体免疫观察奠定了基础。     

甲3型流感病毒在小白鼠肺部的繁殖

甲,型流感病毒与DEAE-D溶液同时接种鼠肺后,再用DEAE-D溶液进行鼻腔刺激,继续传若干代后,病毒很快呈现对鸡红血球有凝集作用,并使动物死亡。血凝滴度和小白鼠LD40滴度分别达1：64和5．0—6．0。用鸡胚病毒的抗血清进行抑制时,抑制滴度与鸡胚病毒相似,但是不被甲。型和仙台病毒的抗血清所抑制。 用原毒株的灭活疫苗免疫小白鼠,然后用鼠肺病毒加DEAE—D攻击,疫苗表现高的保获效价(用正常尿囊液接种作为对照)。本文讨论了利用DBAE—D刺激所获得的鼠肺病毒进行抗流感病毒的药物筛选。     

不同亚型甲型流感病毒在单个核细胞内复制情况研究

目的研究不同亚型的甲型流感病毒在单个核细胞内的复制情况,探讨其免疫应答机制。方法①细胞培养:复苏A549、MDCK细胞后,用DMEM培养液常规培养;外周血分离得到单个核细胞,用RPMI1640常规培养;②空斑形成试验:用空斑试验检测病毒A/Shantou/169/2006(H1N1)和A/Shantou/602/2006(H3N2)的病毒原始滴度;检测流感病毒感染单个核细胞后的病毒滴度变化。结果流感病毒感染单核细胞后,上清液的病毒滴度下降,36、48、72 h病毒滴度<10 PFU/mL;而其细胞裂解液病毒滴度上升,滴度由9.5×10~4 PFU/mL上升至1.63×10~5 PFU/mL。流感病毒感染淋巴细胞,其细胞上清和裂解液病毒滴度均下降,其中上清液H3N2病毒滴度在48、72 h均<10 PFU/mL;裂解液病毒滴度则由4.8×10~5 PFU/mL下降至1.8×10~3 PFU/mL。结论不同亚型的甲型流感病毒在单核细胞和淋巴细胞中的复制存在差异。单核细胞可以吞噬流感病毒但不能直接灭活流感病毒,而淋巴细胞却可以直接抑制流感病毒的复制。     

Modulation of Immune System Function by Measles Virus Infection: Role of Soluble Factor and Direct Infection

Measles virus infection can result in a variety of immunologic defects. We have begun studies to determine the basis for the lack of immune responsiveness to antigen and mitogen following infection. Here we present data showing that Epstein-Barr virus-transformed B-cell lines infected with measles virus produce a soluble factor that can inhibit antigen-specific T-cell proliferation and inhibit the proliferation of uninfected B cells. The soluble factor was neither interleukin-10, transforming growth factor β, nor alpha/beta interferon. B cells infected with measles virus or treated with the soluble factor were unable to present antigen to T cells in a manner that supported antigen-specific proliferation. This could represent one mechanism of how measles virus limits T-cell expansion. However, we found that once CD4⁺ or CD8⁺ T cells were activated, their cytolytic activity was intact whether infected with measles virus or treated with soluble factor. Thus, while slow to be generated these cytoxic cells could participate in viral clearance.     

Measles: immune suppression and immune responses

Measles is a highly contagious viral disease that remains the leading vaccine-preventable cause of child mortality worldwide. Deaths from measles are due largely to an increased susceptibility to secondary bacterial and viral infections, attributed to a prolonged state of immune suppression. Several abnormalities of the immune system have been described, including changes in lymphocyte number and function, shifts in cytokine responses, immunomodulatory effects of interleukin-10, down regulation of interleukin-12, impaired antigen presentation, and altered interferon alpha/beta signaling pathways. Although the current vaccine is very effective, knowledge of the molecular basis of the immune responses to measles virus could contribute to the development of a safer, more immunogenic measles vaccine. However, the safety of new measles vaccines must be carefully investigated, as two measles vaccines have resulted in unintended immunologic consequences: atypical measles following administration of the formalin-inactivated measles vaccine and increased mortality in girls following administration of high-titer measles vaccines.     

Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

Synthesis of the measles virus nucleoprotein in yeast Pichia pastoris and Saccharomyces cerevisiae

The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology. The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively. Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P. pastoris and S. cerevisiae, respectively. This protein is one of most abundantly expressed in yeast. After purification nucleocapsid-like particles (NLPs) derived from both yeast genera appeared to be similar to those detected in mammalian cells infected with measles virus. A spontaneous assembly of nucleoprotein into nucleocapsid-like particles in the absence of the viral leader RNA or viral proteins has been shown. Compartmentalisation of recombinant protein into large compact inclusions in the cytoplasm of yeast S. cerevisiae by green fluorescence protein (GFP) fusion has been demonstrated. Sera from measles patients reacted with the recombinant protein expressed in both yeast genera and a simple diagnostic assay to detect measles IgM could be designed on this basis.     

A Single Amino Acid Change in the Hemagglutinin Protein of Measles Virus Determines Its Ability To Bind CD46 and Reveals Another Receptor on Marmoset B Cells

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.     

Measles virus nucleocapsid protein protects rats from encephalitis.

Lewis rats immunized with recombinant vaccinia virus expressing the nucleocapsid (N) protein of measles virus were protected from encephalitis when subsequently challenged by intracerebral infection with neurotropic measles virus. Immunized rats revealed polyvalent antibodies to the N protein of measles virus in the absence of any neutralizing antibodies as well as an N protein-specific proliferative lymphocyte response. Depletion of CD8+ T lymphocytes did not abrogate the protective potential of the N protein-specific cell-mediated immune response in rats, while protection could be adoptively transferred with N protein-specific CD4+ T lymphocytes. These results indicate that a CD4+ cell-mediated immune response specific for the N protein of measles virus is sufficient to control measles virus infections of the central nervous system.     

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.     

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys

The chimeric recombinant virus rHPIV3-N(B), a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.