Virology-Introduction and Retroviruses

obligate intracellular parasites that require living cells to replicate

what are viruses?

Russian Dimitri Ivanovsky in 1892

who first suggested the existance/discovered viruses?

either DNA or RNA

what is a viral genome?

protein shell enclosing the genome

what is a viral capsid?

subunit of capsid

what is a viral capsomere?

capsid with enclosed genome

what is a viral nucleocapsid?

in some viruses; membrane surrounds nucleocapsid

what is the viral envelope?

the complete, infective form of a virus outside a host cell

what is a virion?

assembly→release→maturation→attachment→penetration→uncoating→replication→gene expression→repeat

what is the viral infectious mechanism?

• based on geographical location—St. Louis encephalitis virus

• Body site—enteroviruses

• mode of transmission—arboviruses (arthropod borne) transmitted by arthropod vectors

what is the historical classification/nomenclature of viruses?

• type of nucleic acid and configuration—DNA or RNA: single stranded/double stranded

• strategy of viral replication—reverse transcriptase

• structure of the virion—symmetry, presence of envelope, diameter of capsid, number of capsomers

what is the current classification/nomenclature of viruses?

• mucous membranes—respiratory, GI, and genital tracts

• skin—breaks, trauma, and infections

• inhalation

what are the major viral routes into the body?

inhalation

what is the most common viral route into the body?

localized or systemic

how can a body site infection appear?

serology

what was the primary means of diagnosing viral infections until mid-1970s?

cell culture, antigen detection, and nucleic acid detection

what replaced serology?

• viruses that are difficult to grow

• detection in acute phase specimens or 4 fold rise in titer

what is serology still used for?

identify viruses by size and shape

what is the function of electron microscopy?

10⁵ to 10⁷ viruses

how many viruses are needed for identification using electron microscopy?

non-cultivable viruses

what is electron microscopy useful for?

initial identification of the pathogen

what does electron microscopy have an important role in?

infected cells show cytoplasmic or nuclear inclusions containing clusters of viruses or viral material

what is seen on a cytology exam?

• host cells are stained with dyes and observed for characteristic morphology

• identify herpes simplex virus—multinucleated cell

what is the Tzanck prep during a cytology exam?

• staining is more sensitive than dye-based staining

• DFA for HSV

what is the fluorescence-labeled antibody test during a cytology exam?

• immunofluorescence

• enzyme immunoassay

• latex agglutination

• rapid immunochromatographic membrane assay

what are methods of antigen detection of viruses?

• silicon wafers coated with antibody + virus

• produce reflection change in water

what occurs during optical immunoassay for viral antigen detection?

• viral antigens that agglutinate RBCs

• guinea pig RBC + infected cells = RING RBCs around infected cells

• virus identified using fluorescent-labeled antibody

what occurs during hemadsorption for viral antigen detection?

screening

what is viral antigen detection used for?

antigen detection methods have lower sensitivities

in general, how does antigen detection assays compare to cell culture, IFA, or other techniques that amplify virus or viral components?

cell culture, IFA, or molecular testing

what is used to confirm negative results antigen detection results?

cell culture

what is the “gold standard” in detecting viruses?

living cells that are kept moist and supplied with nutrients by keeping them continuously immersed in cell culture medium

what does cell culture require?

usually tissue from animal; divides only once or twice

what is primary cell culture?

divide but limited to 50 generation (human neonatal lung or rabbit kidney)

what is diploid cell culture?

capable of indefinite passages (cancerous)

what are continuous cell cultures?

physical changes in appearance of cells due to presence of viral growth

what is cell culture cytopathic effect?

distinct CPE’s

what do viruses have?

• medium is removed from monolayer of cells

• specimen is inoculated

• fresh medium is added

• cell culture is examined for CPE using inverted microscope—examined for days to weeks

what characterizes conventional cell culture?

• microscope with its light source and condenser on top

• objectives and turret are below the stage 

• useful for observing cells on the bottom of a container

what characterizes inverted microscope for cell culture?

• miniaturized cell culture technique

• cells grown on coverslip in small vial with medium; specimen is added to vial and centrifuged

• shell vial incubated 24-48 hours

• monolayer of cells processed for viral antigen before development of CPE

• advantage is speed—viruses detected within 1-2 days

what characterizes the shell vial method of cell culture?

aseptically collected body fluids, tissue, swabs

how should specimens be collected for viral detection?

3-7 days

when should specimens be collected?

• usually specimens other than body fluids

• contains antimicrobial agents to inhibit bacterial and fungal growth

• all viral specimens should be kept on ice, processed immediately or refrigerated

how should viral specimens be transported?

• HIV

• HTLV

what are the retroviruses?

• retro = backward

• RNA virus with reverse transcriptase

• transcribes RNA into DNA (can integrate into host cell)

how did retroviruses get that name?

• HIV-1

• HIV-2

what are the types of HIV?

world wide distribution

what is the distribution of HIV-1?

most cases from West Africa

what is the distribution of HIV-2?

Acquired Immunodeficiency Deficiency Syndrome (AIDS)

what does HIV cause?

• direct contact with blood or mucosa

• cell associated so fewer viruses are found in cell-free plasma than whole blood

• even fewer in saliva, tears, urine, or milk

how is HIV transmitted?

Acute Retrovial Syndrome

• mononucleosis-like

• may occur 1-6 weeks after exposure to virus

• lymphadenopathy, fever, rash, sore throat, fatigue, and neurological manifestations

• may last few days to couple of weeks

• 50-70% of patients

what characterizes HIV Stage 1?

Clinical Latency

• asymptomatic

• active viral production—10 billion viral particles made and eliminated daily

• lymphopenia

  • CD4 cells high turnover

  • can last for years—can transmit to others

• antibody usually present within few weeks after infection

• persistent lymphadenopathy

what characterizes HIV Stage 2?

AIDS

• yeast, fungi, cancers, bacteria, and parasites

• HIV to AIDS

  • developed countries

what characterizes HIV Stage 3?

• decrease T helper cells

  • impair humoral and cellular immunity

  • can also invade macrophages

what is the effect of HIV on the immune system?

>500/µL CD4 (<200/µL →AIDS)

what are the normal values for T helper cells?

spherical, unique 3-layer

• envelope

• matrix

• core (or capsid)

what is the HIV structure?

p24

what is an HIV antigen?

• part of the core

• diagnostically valuable in early infection—precedes appearance of antibody be ~6 days

• becomes undetectable as antibody is produced

what characterizes HIV antigen p24?

• Serology

• CD4 Cell Count

• Rapid Testing

• Cell Culture

what are the HIV Laboratory Tests?

ELISA

what serology test is used for initial HIV screening?

Western Blot

what serology test is used to confirm HIV?

determines degree of immunity and monitors effects of therapy by flow cytometry

what is the purpose of the CD4 Cell Count when testing for HIV?

antibody detection

what is the mainstay of diagnostic testing for adults with HIV?

typically serum or plasma

what are the specimens of HIV antibody detection?

EIA

what is the most common HIV antibody screening test?

can detect antibodies and antigen (p24)

what can EIA with 4th generation detect?

antibodies to HIV-1 and HIV-2

what do most EIA methods detect?

• high sensitivity/specificity in high risk groups

• ~10% of positives are true positives in low risk group

• false negative: collected prior to seroconversion

what characterizes false antibody detection reactions?

• nitrocellulose paper containing HIV antigen is reacted with patient serum, wash

• add enzyme-labeled anti-human antibody, wash, then add substrate

• controls: +, weakly +, and negative

• interpretation of results

  • based on patterns

what is the HIV Western Blot?

at least two of the following bands are present:

• p24

• gp41

• gp120/gp160

when is a positive HIV result reported with Western Blot?

• EIA for p24 antigen

• molecular biology methods—variety

  • detect HIV RNA or DNA

  • used to detect viral load

  • viral load and CD4 count used follow treatment

what is the direct viral detection of HIV?

• provides result and counseling during the initial visit

  • overall sensitivity and specificity >99%

  • not positive for 3-5 weeks after exposure

  • should be confirmed with follow-up test

• confirmatory

  • IFA

what characterizes rapid screening of HIV?

peripheral blood mononuclear cells—look for reverse transcriptase or p24

what is used instead of culture for HIV?

• the “Cocktail”

• antiretrovirals

• highly active antiretroviral therapy

what is the treatment of HIV?

associated with adult T cell leukemia (endemic in southern Japan, Caribbean and Africa)

what characterizes HTLV-1?

not associated with particular disease—role has not been clearly defined

what characterizes HTLV-2?

serology—ELISA method of choice

what is the diagnosis of HTLV?