MYCV Lab - week 10 - Cultivation-of-viruses part 2 PDF

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Week 10- CULTIVATION OF VIRUSES PDetection of virus growth in cell culture  Can be detected by the following methods o Cytopathic effect o Metabolic inhibition o Hemadsorption o Interference o Transformation o Immunofluorescence o Detection of virus-specific nucleic acid o Detection of enzymesCytol...

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Week 10- CULTIVATION OF VIRUSES P2 Detection of virus growth in cell culture  Can be detected by the following methods o Cytopathic effect o Metabolic inhibition o Hemadsorption o Interference o Transformation o Immunofluorescence o Detection of virus-specific nucleic acid o Detection of enzymes

Cytopathic Effect  Morphological changes in cultured cells o ‘cytopathic effects’ (CPE) / ‘cytopathogenic viruses.’  Presumptive identification of a virus isolated from a clinical specimen. Main Types of CPE  o Rounding of cells - picornaviruses o Cell necrosis and lysis – Enteroviruses o Syncytium formation - measles,respiratory syncytial virus, human immunodeficiency virus (HIV) o Discrete focal degeneration - Herpes virus o Rounding and aggregation - Adenovirus

Cytology and Histology  Viral inclusions o components in an infected cell or abnormal accumulations of cellular materials resulting from virus-induced metabolic disruption.  Intracellular structures formed by aggregates of virus or viral o Single o Syncytial cells aggregates of cells fused to form one large  cell with multiple nuclei.  Inclusions from this virus detected by histology o CMV o Adenovirus o Parvovirus

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o Papillomavirus o molluscum contagiosum virus Inclusions characteristic of measles and rabies viruses o detected by examining stained tissue Rabies virus inclusions in brain tissue - negri bodies. less sensitive than culture but are especially helpful for viruses that are difficult or dangerous to isolate in the laboratory o o parvovirus o o rabies virus

A, Pap-stained smear showing multinucleated giant cells typical of herpes simplex or varicella-zoster viruses. B, Hematoxylin and eosin (HE)–stained lung tissue containing intranuclear inclusion within enlarged

C, HE-stained lung tissue containing epithelial cells with intranuclear inclusions characteristic of adenovirus. E, Pap stain of exfoliated cervicovaginal epithelial cells showing perinuclear vacuolization and nuclear enlargement characteristic of human papillomavirus infection. F, HE-stained epidermis filled with molluscum bodies, which are large, eosinophilic, cytoplasmic inclusions resulting from infection with molluscum contagiosum virus.

G, HE-stained cells infected with measles virus. H, HE-stained brain tissue showing oval, eosinophilic rabies cytoplasmic inclusion (Negri body).

D, HE-stained liver from stillborn fetus showing intranuclear inclusions in erythroblasts (extramedullary hematopoiesis) resulting from parvovirus infection.

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Phenomenon for which a cell infected by a virus becomes resistant toward a second outcoming infection by a superinfectant virus. Rubella virus o does not produce cytopathic changes, although they multiply within the cell

Transformation  Tumor forming (oncogenic) viruses  Growth appears in a piled-up fashion producing microtumors o Herpes viruses o Adenoviruses, o Hepadnaviruses, o Papovaviruses o Retroviruses Immunofluorescence  Can be stained by fluorescent conjugated antiserum  Examined under the UV microscope for the presence of virus antigen  Gives positive results earlier than other method

Metabolic Inhibition  Cell metabolism is inhibited and there is no acid production  Made out by the color of the indicator (phenol red) incorporated in the medium Hemadsorption  Addition of guinea pig erythrocytes to the cultures o Influenza o parainfluenza viruses o Erythrocytes will adsorb onto the surface of cells if the viruses are multiplying in the culture  Becomes positive before cytopathic changes are visible and in some cases occurs in the absence of cytopathic effects

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Direct immunofluorescent testing o use of a labeled antiviral antibody; the label is usually fluorescein isothiocyanate (FITC) - layered over a specimen suspected of containing a homologous virus o generally more rapid and specific but less sensitive o best suited to large quantities of virus are suspected or when high-quality, concentrated monoclonal antibodies are used

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Indirect immunofluorescent procedure o two-step test in which unlabeled antiviral antibody is added to the slide, followed by a labeled (FITC) antiglobulin that binds to the firststep antibody bound to virus in the specimen. increased sensitivity of indirect o immunofluorescence results from signal amplification that occurs with the addition of the second antibody o decreases specificity by increasing nonspecific background fluorescence o used when lower quantities of virus are suspected, such as detection of respiratory viruses in specimens from adult patients A, RSV-infected RMK (rhesus monkey kidney) cells at 400×, stained with Light Diagnostics RSV MoAb. Fluorescence is seen in the cytoplasm and associated with syncytia. Cytoplasmic staining is often punctuate with small inclusions.

Interference  Growth of a non-cytopathogenic virus is tested by the subsequent challenge with a known cytopathogenic virus o Growth of the first (non-cyto) will inhibit infection by the second virus (cyto) by interference

B, HSV I: HSV I infected Vero cell control slide 200×. Stained with Pathfinder HSV 1 MoAb DFA assay. Fluorescent staining is cytoplasmic.

C, Influenza B infected RMK cells at 400×. Stained with Light Diagnostics Influenza B MoAb. Fluorescence is nuclear, cytoplasmic, or both. Nuclear staining is uniformly bright and the cytoplasmic staining is often punctuate with l large inclusions. D, Herpes Simplex II infected A549 cells at 200×. Stained with Pathfinder HSV II MoAb DFA assay. Fluorescence may stain the cytoplasm, the nucleus,

Electron Microscopy Distinctive appearances and can be detected by electron  microscopy of ultra thin sections of infected cells

E, HSV II Infected A549 cells at 200×.

F, HSV II Infected A549 cells at 400×. Picture shows the multinucleated “giant” cells characteristic of HSV II CPE (cytopathogenic effect).

Detection of Virus-specific Nucleic Acid Molecular-based assays, such as polymerase chain reaction   Provide rapid, sensitive, and specific methods of detection A, Rotavirus.

B, Adenovirus

C, Norwalk agent virus.

D, Coronavirus

G, E, Negatively stained Herpes preparation of JC simplex virus. virus in brain tissue

F, Measles virus.

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Viral assay Distinctive appearances and can be detected by electron microscopy of ultra thin sections of infected cells Total virus particles o Electron microscopy o Hemagglutination Infectious virion assay o Quantal assays o Quantitative infectivity assay

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Detection of Enzymes  Identified by detection of viral enzymes, such as reverse transcriptase in retroviruses, in the culture fluid

Total Virus Particles Electron Microscopy  By simple negative staining, the virus particles in a suspension can be counted directly under the electron microscope.

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The virus suspension can be mixed with a known latex particles. The ratio between particles under the electron microscope gives an indication of the virus count.

Hemagglutination  A convenient method of quantitation is the determination of hemagglutination titers with hemagglutinating viruses.  Hemagglutination is not a very sensitive indicator of the presence of small amount of virus particles.  approximately 107 influenza virions are required to produce macroscopic agglutination.

 Infectious Virion Assay o Quantal Assays  Quantal assays only indicate the presence or absence of infectious viruses.  Quantal assays of infectivity can be carried out in animals, eggs or tissue culture for those viruses that do not form plaques  endpoints used for infectivity titration  death of the animal  production of hemagglutinin in allantoic fluid  appearance of CPE in cell cultures  titer is usually expressed as the 50 percent infectious dose’ (1D50) per mL, which indicates the highest dilution of the inoculum that would produce an effect in 50% of animals, eggs or cell cultures inoculated o Quantitative Infectivity Assay Quantitative assays measure the actual  number of infectious particles in the inoculum. Two methods are available   plaque assay in monolayer cell culture  pock assay on chick embryo CAM

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Plaque Assay  Viral suspension is added to a monolayer of cultured cells in a bottle or petri dish nallowing time for absorption,  Removed and replaced with a solid agar gel to prevent virus spreading throughout the culture  Each infectious viral particle gives rise to a localized focus of infected cells that can be seen with the naked eye. - Known as

‘plaques’ and each plaque indicates an infectious virus

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Pock Assay Herpes and vaccinia, o form pocks when inoculated onto the chorioallantoic membrane of an embryonated egg. o Such viruses can be assayed by counting the number of pocks formed on cam by appropriate inocula of virus.

HSV Grow rapidly in different cell lines o MRE-5 o Human fibroblast o Vero cell o Hep-2 o Mink lung o PMK Grow at CPE within 24 hrs

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Rapid in CPE

CMV  

HSV like Grow in CPE slowly o Diploid fibroblast

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Grow at different cell type o Diploid fibroblast o A549 o Vero cell

V2V

Entero 

Adeno  

Small, round o PMK o Diploid fibroblast o Human embryonal (RD) o Rhabdomysarcoma cell  A549 Round large Cluster of grapes o Diploid fibroblast o PMK o Hep-2 o A549

Rebulla  

Do not produce CPE Challenged

RT-PCR  

Target amplification and detection Simultaneous in the same tube

Conventional PCR  Separate amplification  Separate detection Electron microscopy  Distinctive appearance  Gastroenteritis viruses  Encephalitis causing virus...