复旦大学：《医学微生物学 Medical Microbiology（MBBS）》课程讲义_理论课_Lecture15-Virus Genetics & Evolution

Genetics Evolution of viruses Youhua xie(谢幼华) MOH&MOE Key Lab of Medical Molecular Virology Shanghai Medical College, Fudan University 复旦大学上海医学院 分子病毒学教育部/卫生部重点实验室 yhxie@fudan.edu.cn

Genetics & Evolution of Viruses Genetics & Evolution of Viruses Youhua Xie Youhua Xie (谢幼华 ) MOH&MOE Key Lab of Medical Molecular Virology Sh h i M di l C ll F d U i it Shang h ai M edical C ollege, F u dan U niversit y 复旦大学上海医学院 分子病毒学教育部 /卫生部重点实验室 yhxie@fudan.edu.cn

Basic Terms Used in viral Genetics Genotype Genetic makeup of a virus a subpopulation of viruses of a virus species that share more homology with each other in nucleic acid sequence and/or amino acid sequence than with other subpopulations Patients infected with genotype b and c of hepatitis b virus respond to the treatment quite differently

Basic Terms Used in Viral Genetics z Genotyp e ¾ Genetic makeup of a virus ¾ A subpopulation of viruses of a virus species that share more A subpopulation of viruses of a virus species that share more homology with each other in nucleic acid sequence and/or amino acid sequence than with other subpopulations “Patients infected with genotype B and C of hepatitis B virus respond to the treatment quite differently

Basic Terms Used in viral Genetics Phenotype Observable characteristics or traits, produced by genotype as well as the influence of environmental factors and the interactions between the two ● Genome The sum of the genes of a virus ● Wild-type virus A virus from which a mutant virus is made and compared with Mutations. mutant viruses Field isolates or primary isolates

Basic Terms Used in Viral Genetics z Phenotyp e ¾ Observable characteristics or traits, produced by genotype as well as the influence of environmental factors and the interactions between the two the two z Genome ¾ The sum of the genes of a virus z Wild-type virus ¾ A virus from which a mutant virus is made and compared with z Mutations, mutant viruses z Field isolates or primary isolates

Detection of viruses o Detection methods that are independent on viral infections Do not distinguish between infectious and particles Viruses detected by hemadsorption of human or animal erythrocytes(hemagglutination assay Detection of viral antigens [immunofluorescence; enzyme immunoassay(ElA); radioimmunoassay(RIA) Detection of viral nucleic acids PCR-based detection of unkown viruses nucleic acid n tests(NAATS) Identification of emerging viruses(genechip; high-throughput sequencing) Observation under electron microscope [direct or immune electron microscopy (IEM)

Detection of Viruses z Detection methods that are independent on viral infections Detection methods that are independent on viral infections. Do not distinguish between infectious and noninfectious viral particles ¾ Viruses detected by hemadsorption of human or animal erythrocytes (hemagglutination assay ) ¾ Detection of viral antigens [immunofluorescence; enzyme immunoassay (EIA); radioimmunoassay (RIA)] ¾ D t ti f i l l i id Detection of viral nucleic acids „ PCR-based detection of unkown viruses [nucleic acid amp ( )] lification tests (NAATs)] „ Identification of emerging viruses (genechip; high-throughput sequencing) ¾ Observation under electron microscope [direct or immune electron microscopy (IEM)]

Hemagglutination Assay t buffer virus dilution RBc(approx. 105) ◎ 1:4 + virus|oo° buffer 1:2048 1:4096 Hemagglutination assay

Hemagglutination Assay + buffer + virus dilution + RBC (approx. 10 5) 1:4 … + virus + buffer … … 1:2048 1:4096 Hemagglutination assay

Immunofluorescence Ab DAPI HBSAg Overlay fluorescin Mock A Vild-type HBV

Immunofluorescence Ab fluorescin DAPI HBsAg Overlay Mock Ag Wild-type HBV

Enzyme-Linked Immunosorbent Assay(ELISA) and transfer 0.1ml Virus preparation Virus concentration Substract Substract Substract Substract Secondary antibody conjugate Primary antibody conjugate g Captue antibody Direct In Direct Sandwich Competitive

Enzyme-Linked Immunosorbnent Assay (ELISA) Virus preparation Virus concentration Substract Substract Substract Substract Secondary antibody conjugate Ag Primary antibody conjugate Captue antibody Inhibitor Ag Direct InDirect Sandwich Competitive

Qualitative quantitative PCR Control Experimental assay at 25 cycles Control Positive Negative 2.5 82.0 1.5 1.0 0.5 0 10 40 50 Amplification cycles Computer calculates ratio of intensities

Qualitative & Quantitative PCR

Genechip Reverse In vitro Transcription Biotinylated -inear/ Exponental 885如丛 scanner Automated FluidIcs - e http://www.keywordpicture.com/keyword/trizol%20protocol/

Genechip http://www.keywordpicture.com/keyword/trizol%20protocol/

Electron Microscopy Lesion cun An4+ Viral particles of hbv http://www.purdue.edu/rem/rs/sem.htm

Electron Microscopy Viral particles of HBV http://www.purdue.edu/rem/rs/sem.htm