第14章 病毒感染的检查方法与防治原则

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cytopathic effect, CPE
③ inclusion body formation
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Detection of Virus Infected Cells---Not produce a CPE
➢ Adsorption Erythrocytes to Infected Cells:
Restriction endonuclease can cleave viral DNA at a specific nucleic sequence, and are able to produce a set of DNA fragment of viral genome.
They can be separated by gel electrophoresis give a characteristic result for the virus DNA.
Hemagglutination Assay
➢ Interference: When two different viruses infect the
cells. First non-CPE virus may be inhibit the replication of second virus that induce CPE in infected cells.
CPE including: * morphologic changes: cell lysis, necrosis, and cytoplasm
vacuolization.
* fusion of cells form multinucleated giant cells:
Measles virus, CMV, respiratory syncytial virus；
2. 蚀斑形成试验
（plaque formation test）
3. 50％组织细胞感染量 （50％ tissue culture infectious dose, TCID50）
感染性病毒量
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PLAQUE ASSAY
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PLAQUE ASSAY
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* inclusion body formation: Rabies virus, measles virus.
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cytopathic effect, CPE
respiratory syncytial virus
measles virus
② fusion of cells form multinucleated giant cells
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(3) 传代细胞培养：来自癌细胞、自发转 化或突变细胞。
特点：①对多种病毒敏感性高 ②繁殖能力高，传代时间长 ③有致癌危险，不能用于疫苗生产
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病毒的鉴定 1．病毒在培养细胞中增殖的指标
virus
normal cells
multiply
produce CPE
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Detection Viral nucleic acid
Nucleic Acid Probes-sensitive and specific The labeled (with either radioactive substance or a dye) specific methods
病毒感染的检查
EM: virus particle
LM: inclusion body
V.particles
Inoculation and
Diagnosis
Virus
Viral Ag.
Cultivation
Viral DNA/RNA
Viral enzymes
Specific Ab.
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PCR----DNA replication
5’
3’
3’
5’
5’
3’
3’
5’
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PCR----DNA replication
5’
5’ 3’
5’ 3’
3’ 5’
5’ 5’ 3’
5’ 3’ 5’ 3’
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3’ 5’ 3’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 5’
3’ 5’
➢ Metabolic Changes: Such as the pH reduces.
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Hemagglutination Assay
病毒包膜表面血凝素(HA) ＋
红细胞表面HA受体
2020/8/16 Influenza virus hemagglutinin trimer
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3’ 5’ 5’
3’
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Detection Viral nucleic acid
Polymerase chain Reaction (PCR)
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Detection Viral Antibody---Serologic Tests
(1) Neutralization: (2) Complement Fixation (CF) Tests (3) Hemagglutination Inhibition (HI) Test (4) Immunodiffusion Test (5) Enzyme-Linked Immunosorbent Assay (ELISA)
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酶联免疫吸附试验（ELISA,间接法）
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Detection Viral Antigen
Some viral infections can be detected by making a specific antibody against the virus: The antibody is conjugated to a tag such as a fluorescent dye (IFA) and Radioimmunoassay(RIA)
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Quick diagnosis of viral infection
病毒颗粒 病毒成分（Ag.或核酸） 早期Ab检测（IgM）
1.病毒形态学检查 电镜免疫电镜：设备、操作复杂，应用受限
光学显微镜：可见大病毒（痘类病毒），
对一般病毒检测意义不大
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（二）病毒蛋白Ag检测
酶联免疫吸附试验(ELISA) 免疫荧光测定(IFA) 放射免疫测定(RIA) 蛋白质印迹(WB)
hybridization
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④ in situ hybridization
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Kary Mullis
1985 2K02M0/u8l/l1i6s, colleagues, Cetus, Perkin-Elmer
invention of the polymerase chain reaction (PC4R2)
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2. embryonated eggs inoculation
优点：易管理，不带微生物，成本较低 缺点：操作程序复杂
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3. Tissue or cell culture
分类
单层细胞培养（monolayer cell culture） 悬浮细胞培养（suspended cell culture）
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（三）、检测病毒核酸
核酸电泳 核酸杂交（dot,in situ） 核酸扩增技术（PCR） 基因芯片 基因测序
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Detection Viral nucleic acid
Restriction Endonuclease Analysis (REA):
Hemagglutination Assay----
red cell agglutination test
Hemagglutination Assay
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Hemagglutination inhibition test
鉴定病毒种、型（亚型） 红细胞凝集和血凝抑制试验 病毒＋鸡红细胞＝凝集（血凝试验+）
①dot-blot hybridization ②Southern blot hybridization ③Northern blot ④ in situ hybridization.
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Detection Viral nucleic acid
①dot-blot hybridization ②Southern and ③Northern blot
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一、标本的采集与送检
Collection and transport of specimens
原则基本与细菌相似，但应注意：
1.急性期标本 2.根据感染性质取不同标本 3.标本用“双抗”处理 4.低温快运 5.血清学诊断采双份血清
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二、病毒的分离与鉴定
Viral isolation and identification
培养基
单层细胞 (原代细胞)
传代培养 单层细胞
(次代细胞)
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(1)原代和次代细胞培养 特点：①对多种病毒敏感 ②生长能力有限, 获取成本高 ③可携带潜伏病毒
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