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VIRUS LESSON 1: INTRODUCTION TO VIROLOGY The smallest particle that may cause harm and disease to human. belong to any kingdom. not a plant or an animal. not a fungi, protist or bacteria. have any cellular structure. Properties of Virus Properties Types of Nucleic Acid Protein Lipoprotein Membrane R...

Description

VIRUS     

LESSON 1: INTRODUCTION TO VIROLOGY



The smallest particle that may cause harm and disease to human. Doesn’t belong to any kingdom. It’s not a plant or an animal. It’s not a fungi, protist or bacteria. Doesn’t have any cellular structure.

   

Virus measures in terms of nanometer using scanning microscope. Size of viruses: 20 – 300 nm Bacteriophage – viruses that infect bacterial cell. Poliovirus –smallest virus recorded clinically measures 25 nm. Poxvirus – largest virus measures 250 nm x 350 nm.

a. b.

Can be DNA or RNA Can be Double Strand or Single Strand 

 

Properties of Virus



DNA – DS (Hepatitis B virus, Varicella – zoster virus, Human papilloma virus, JC virus) DNA – SS (Parvovirus B – 19) RNA – SS (Influenza virus, HIV, Ebola virus) RNA – DS (Rotavirus)

SHAPE OF A VIRUS c. Properties Types of Nucleic Acid Protein Lipoprotein Membrane Ribosomes Mitochondria Enzymes Multiplication by Binary Fission/Mitosis

Virus   



Virus DNA or RNA, never both Few Present or few to some viruses Absent Absent Absent or Few

Cell RNA and DNA Many

1.

Spherical – majority of the virus. Ex. HIV, Adenovirus, Influenza virus, Rotavirus

Many

2.

Rod – shaped Ex. Bacteriophage

No

Yes

Present Present Many

Is an infectious agent made up of nucleic acid (DNA or RNA) wrapped in a protein coated called a capsid. No nucleus, organelles, cytoplasm or cell membrane. Are parasites – an organism that depends entirely upon another living organism (a host) for its existence in such a way that it harms that organism. Obligate intracellular parasite.

3.

Bullet – shaped Ex. Rabies virus

4.

Brick – shaped Ex. Smallpoxvirus

5.

Filament Ex. Ebola virus, Marburg Virus

Can be single molecule or segmented o DNA – always a single molecule o RNA – can be separated or a single molecules

Ex. Multiple/Segmented – Influenza virus, Rotavirus d.

Linear or Circular(arrangement of nucleic acid)

* A virus is always haploid (23 chrom.) except Retrovirus (causes HIV), which is diploid (46 chrom.)

Capsid Protein coat that surrounds the nucleic acid and protects the gene. ANATOMY OF VIRUS Identifies the specificity of the virus. Can be detected serologically. Virion – entire/complete particleof a virus Composed of repeating subunit called capsomere. 1. Nucleic Acid–innermost part; determine genome Two Possible Symmetry of Virus(Capsid structure) 6.

Tadpole – shaped Ex. Bacteriophage

    

Poliovirus Coxsackie virus Rotavirus Hepatitis A virus Echovirus (Enterocytopathic human orphan virus)  Norwalk virus

a. Icosahedral –Capsomere arrange in 20 triangles that in symmetry figure with the approximate outline of the sphere. (NAKED) b. Helical – Capsomere arrange in hallow coil that arrange in rod shaped. (ENVELOPED – either icosahedral or helical) 3.

Nucleocapsid– nucleic acid +capsid

4.

Enveloped  Lipoprotein membrane compose of lipid derive from host cell and specific protein.  Confirms the instability  Once the virus has envelope they are not stable/unstable very susceptible to extreme conditions such as heat, dryness, exposure to detergent, extreme heat and lipid solvents like alcohol and ether  Contains glycoprotein spikes

b.

5.

c.

Naked  Without lipoprotein/envelope  Very resistant to environmental factors  Because of their stability, they are typically transmitted by fecal-oral route

7.

Protein Matrix/Matrix Protein – mediates the interaction between the nucleocapsid and the envelope.

d.

Glycoprotein Spikes – spike – like protection found in surface of envelope used for attachment.

Envelope/Naked can be Classify by Mode of Transmission a.

Feca – oral NAKED

e.

1. 2. 3. 4. 5.

Direct Transmission ENVELOPED  Respiratory Droplets:  Varicella – zoster virus (causes chickenpox n children)  Rubella virus  Influenza virus  Measles virus  Mumps virus Sexually Transmitted ENVELOPED  Human immunodeficiency virus (HIV)  Herpes simplex virus o Type I – blisters in mouth o Type II – genital area  Cytomegalovirus 

6.

Rules In Naming:

Except Human papilloma virus(HPV)– doesn’t have envelope (Naked virus)

Insect Bite ENVELOPED  Dengue virus  Yellow fever virus  West Nile virus  Chikungunya virus Animal Bite ENVELOPED  Rabies virus

Order – Single word with suffix “virales”. Family – single word with suffix “viridae”. Subfamilies – single word with suffix “virinae”. Genus – single word with suffix “virus” Species – 2 or more words with “virus”.

Measles virus

HIV

Dengue virus unassign ed

Mononegaviral es

unassigned

Paramyxovirida e

Retroviridae

Flavivirid ae

Paramyxovirina e

Orthoretrovirin ae

None

Genus

Morbilivirus

Lentivirus

Flavivirus

Speci es

Measles virus

HIV

Dengue virus

Order Famil y Subfamili es

SEVEN (7) ORDER OF VIRUS 1. 2. 3. 4. 5. 6. 7.

Caudovirales Herpesvirales Ligamenvirales Mononegavirales Nidovirales Picornavirales Tymovirales

VIRAL REPLICATION Specific Events during growth cycle of a virus:

2. A.

B. Middle Events

Early Events 1. Attachment  Protein part attached to the surface of the receptor site of the host cell in a weak and non-covalent bonding  Lock and Key Concept Viruses with Identified/Own Receptor Site:

4.

Gene Expression  synthesis of mRNA  most important

C.

Late protein – structural type of protein virus after gene replication; capsid.

Late Events 6.

Factors that affect the synthesis of mRNA: a. Types of Nucleic Acid DNA (nucleus) except Poxviridae– gene 7. expression in cytoplasm because they o have polymerase. o RNA (Cytoplasm)except Influenza virus, HIV, Hepatitis D virus (nucleus) because of reverse trascriptase

Assembly  Assemble together to make a virion – complete particle of virus.

o Virus Poliovirus Epstein – Barr

Target Cells Epithelial B cellcells Many cells

Herpes simplex virus type 1 R Rabies virus (colds)

Acetylcholine (intercellular – receptor adhesion molecule) Epithelial Sialic Acid cells Erythroid Erythrocyte P precursors antigen

Penetration  Will go inside/enter the host cell that will undergo several mechanism for penetration  

3.

Fibroblasts growth factor receptor

Neuron cells

Influenza A virus Parvo B19 virus

2.

Receptor Ig – like C3D Complement receptor/CD155

Mechanisms/Type of Penetration: If Envelope virus – cell fusion/plasma membrane fusion o If Naked – Endocytosis (entry in the vacuolated part of the cell

Uncoating o o

Removing of protein coat to free/release the nucleic acid In low pH of cytoplasm

b.

5.

Release  Based on the type of virus. 

Polarity o + polariry– using RNA itself o –polarity – carries its own polymerase to convert o polymerase to convert

Gene replication  Copying of the segment of nucleic acid into different copies/multiple pieces.

Two Types of Protein Synthesized 1. Early Protein – necessary to replicate (enzyme nonstructural elements); produce before gene replication.



If Envelope – budding/reverse phagocytosis/or in lipoprotein of the cell such as cell membrane (nuclear membrane in Herpes virus). If Naked –lysis/rupture of cell membrane.

+SS RNA

- SS RNA

Poliovirus Rhinovirus Corona virus Retroviridae Flaviviridae Togaviridae Caliciviridae

Rabies virus Influenza virus Paramyxoviridae

DS RNA (ambisense ) + or Reoviridae

Filoviridae Bunyaviridae Arenaviridae Deltaviridae

 Eclipse Period –time or period where there is no virus detected inside a cell.

 0 – 2 hours  Eclipse period – no virus detected inside a cell (uncoating to assembly).  10 hours finished their replication.  2 hours  Fast Replication – Picornavirus  Slow Replication - HIV Cytopathic Effect The functional or morphological rearrangement of host cell infected by a virus. Most important initial diagnosis for viral infection. Not all causes CPE Start at the end of latent period. Latent Period time from the onset of infection to the appearance of virus extracellularly starts from the appearance of virus Examples of Cytopathic Effect of Viral Infection  Nuclear shrinking (pyknosis)  Proliferation of nuclear membrane  Vacuoles in cytoplasm  Syncytia (cell fusion).  Margination and breaking of chromosomes.  Rounding up and detachment of cultured cells.  Inclusion bodies  Negri body – found in patients with Rabies infected such as brain cells.

Classification of Clinically Significant Viruses  Virion morphology (shape, size)  Physiochemical properties of the virion

 

(lipid, enzyme) Virus genome properties (RNA/DNA, ss/ds, linear/circular) Virus protein properties (Icosahedral/helical/complex).

DNA VIR VIRUS US F FAMIL AMIL AMILY Y  Parvoviridae o Parvovirus B19 

Polyomaviridae o JC polyomavirus o Bkpolyomavirus



Papillomaviridae o HPV



Adenoviridae o Human Adenovirus



Hepadnaviridae o Hepatitis B virus



Herpesviridae o Cytomegalovirus o Herpes simplex virus o Human herpes virus 6, 7, 8 o Epstein – barr virus o Varicella – zoster virus,

o o o o 

Hepeviridae o Hepatitis E virus



Caliciviridae o Norwalk virus (causes diarrhea)



Reoviridae o Colorado Tick fever virus o Rotavirus



Flaviviridae o Dengue Virus o Japanese encephalitis virus o West Nile virus o Hepatitis C virus o Yellow Fever virus



Togaviridae o Rubella virus o Chikungunya virus



Retroviridae o HIV o Human T cell Leukemia virus I, II



Orthomyxoviridae o Influenza A, B, C virus Paramyxoviridae o Mumps virus o Measles virus



Poxviridae o Smallpox virus o Molloscumcontagiosum virus o Vaccinia virus RNA VIR VIRUS US F FAMIL AMIL AMILY Y  Picornaviridae o Coxsackie virus

Hepatitis A virus Echo virus Rhinovirus Polio virus



o o

Respiratory syncytial virus Parainfluenza virus

Serologic procedures to detect a vise in antibody titer or the presence of IgM. Detection of viral antigen in blood or body fluids. Detection of viral nucleic acid in blood or the patient’s cells.



Trypticase Soy Broth or Buffer Normal Saline Solution:for avoiding drying of specimen



Have gelatin, albumin and serum: for protection of less viable virus

Specimen Collection, Handling and Transport  Aseptically collection  Delayed: 1 – 6oC less than 24 hours  Long storage: freezer at – 70oC(cryoprecipitate)  Collect by Dacron or Rayon  Calcium alginate – type of swab that is not allowed to be used because it may damage or inactivate the virus  Specimen that are aspirated during collected for cell culture and does not need transport medium: o Blood o CSF o Pleural fluid o Synovial fluid o Pericardial fluid o Urine o Bone marrow aspirate



Has antibiotics and antifungal: to inhibit the growth of bacteria and fungi

3. 4.





Rhabdoviridae o Rabies virus Filoviridae o Ebola virus o Marburg virus



Coronaviridae o Corona virus (SARS)



Arenaviridae o Lymphocytic Choriomeningitis virus o Lassa Fever virus



Bunyaviridae o California encephalitis virus o Lacrosse virus o Hantavirus



Deltaviridae o Hepatitis D virus

LESSON 2: LABORATORY DIAGNOSIS, TREATMENT AND PREVENTION OF VIRAL INFECTIONS Laboratory Diagnosis of Viral Infection 1. Identification of the virus in cell culture. 2. Microscopic identification directly in the specimen.

5.

Transport Medium  Viral Transport Medium except for blood, urine and other body fluids.  Specimens that need transport medium which is collected only in swabs or respiratory specimen o Throat o Nasopharyngeal o Vaginal

IDENTIFICATION OF THE VIRUS IN CELL CULTURE Methods of Viral Isolation A. Cell Culture B. Animal Inoculation C. Embryonated egg Inoculation A. Cell Culture  Gold standard (in culturable virus)  Presumptive test

Categories of Preparation: 1.

Primary Cell Cuture derived from an animal cell Rabbit Kidney (RK)or Primary Monkey Kidney (PMK) a.

b. c.

A tissue is treated with enzyme to separate the cells (treated with trypsin). Cells are suspended in culture medium. Normal cells or primary cells grow in a monolayer across the glass or plastic container. Transformed cells

or continuous cell culture do not grow in a monolayer. 2.

Finite Cell Culture – 50 generations Ex. Human Diploid Fibroblast (HDF) HumanNeonatalLung (HNL)

RSV

Continuous Cell Culture  Heteroploid – normal or abnormal tissue Ex. A549 – Human Lung Carcinoma

cell line

1.

HEp2 – Human Laryngeal Carcinoma

Observation of their Cytopathic Effects 1. Change in Shape 2. Change in Size 3. Or Syncytia – type of cell multinucleated giant cell resulted from fusion of 2 or more cells. * HBV – non-culturable * M.leprae – culturable in tissue cells only

HSV

PMK HDF HEp2 RK A549 –

+++ +++ +++ +++

CMV

–

+++

–

–

–

VZV

–

+++

–

–

+/–

Enterovirus

+

+

++

–

+

Cytopathic effect large, rounded cells large, rounded cells foci or rounded cells, possible of syncytia Refractile round cells

+

++

+++

–

++

–

–

–

+++

++

++

in clusters Syncytia Variable (no CPE, or CPE of granular appearance) Large rounded cells in clusters

Techniques employed if there is no CPE:

cell line

Virus

+

Influenza, parainfluenza +++ +/– virus

Adenovirus

3.

+/–

Hemadsorption  Surface RBC adsorption  Applicable only for enveloped viruses  Virus from flask without CPE  put in a reaction well + suspected known antibody = Ag-Ab reaction  Reagent: BLOOD  2 types of glycoprotein spike: o Hemagglutinin– found on the surface of enveloped viruses; will attach to the RBC o Neuraminidase Ex. Mumps Virus, Influenza virus, Parainfluenza virus

2.

Interference  Formation of CPE by a 2nd virus, once it was added in the cell culture Ex. Rubella virus + Coxsackie virus

= CPE

3.

Decreased acid production by infected , dying cells  Culture medium + pH indicator(phenol red) =

(+) YELLOWVirus non infected cells; Metabolism happened or Acid production (–) RED Virus infected cells; Alkaline; No metabolism occurred  Phenol red has a neutral pH

Definitive Tests (Confirmatory Test) – Serological Assay 1.

Complement fixation  Viral Ag + Ab + RBC(indicator reagent) = (+) No hemolysis (–) Hemolysis

2.

Hemagglutination inhibition  Viral Ag + RBC(indicator in reagent w/Ab) = (+) No agglutination (–) Agglutination

3.

Neutralization – viral Ag blocks Ab that will be directed to RBC neutralizing it

4.

Fluorescent antibody assay  Labeled assay  Ag + Ab (reagent) tagged with fluorescein (make use of UV light) = (+) Fluorescence (–) No fluorescence

C. 1.

2.

Radioimmunoassay (RIA) – tagged with radio Enzyme linked immonosorbent assay – tagged with enzyme

Viruses Isolated by Cell Culture Problems with Cell Culture 1. Long period (up to 4 weeks) required for result 2. Often very poor sensitivity, sensitivity Viruses readily

         

CMV Adenovirus Polio virus ECHO virus Mumps virus Coxsackie A virus HSV Influenza virus RSV Parainfluenza virus

3. 4. 5.

Embryonated Egg Inoculation  Common in industries that produces vaccines Inoculations in the different parts of the egg: 1) Choriallantoic membrane inoculation o Rous sarcoma virus o Herpes simplex virus o Pox virus

VZV

Rhinovirus Coxsackie A virus

B. Animal Inoculation  Inoculation in a testing animal

Tzanck Test/ Tszanck Smear A stained smear of cells from the base of skin vesicle used to detect VZV or HSV inclusions Uses Papanicolaou stain, Giemsa stain, Hematoxylin& Eosin stain Detects the multinucleated giant cells and inclusions



Koilocytosis / Oval owl’s eye Alteration of the cell with HPV (have no CPE) as squamous cells with enlarged nucleus surrounded by nonstaining halo seen in Papanicolaou stain (Pap smear) Indication of CMV and HPV infection

3) Yolk sac inoculation o Herpes simplex virus

Measles virus Rubella virus

depends on a large extent on the condition of the specimen Susceptible to bacterial contamination Susceptible to toxic substances which may be present in the specimen Many viruses will not grow in cell culture e.g. Hepatitis B, diarrheal viruses (Norwalk virus), Parvovirus, Papillomavirus



2) Amniotic inoculation o Influenza virus o Mumps virus

Less frequently

    

Ex. Negri bodies/ Purkinje cell – Rabies virus Guarnieri bodies – Smallpox virus Cowdry type A bodies – HSV, VZV

4) Allantoic inoculation o Influenza virus o Mumps virus o New castle disease o Avian adenovirus MICROSCOPIC IDENTIFICATION DIRECTLY IN THE SPECIMEN 3 Procedures: 1.

Light Microscopy  Uses light microscope  Presence of inclusion bodies (cowdry type A bodies)  Possible CPE in light microscope

2.

Ultraviolet Microscopy 

Uses immunofluorescence assay



Adenovirus, Influenza type A & B virus, Measles virus, RSV

Clin. SpecimenSputum, throat swab, bronchial lavage 



3.

Herpes simplex virus, Varicellazoster virus o Cutaneous specimens Skin biopsy, skin lesion Cytomegalovirus o Specimen Blood

Electron Microscopy  Directly seen structural arrangement of the virus  Visualization/ detection of size and shape to identify the virus present  For research purposes  106 virus particles per ml required for the visualization, 50,000 – 60,000 magnification normally used. Viruses may be detected. 

 

Feces  Rotavirus, Adenovirus, Norwalk virus Vesicle fluid ...