lab.1/ virology
viral structure:
viruses are composed of a nucleic acid genome surrounded by a protein coat called a capsid-togather the genome and capsid are referred to as the nucleocapsid. genomes are either rna or dna. viral capsid are composed of many individual subunits called capsomeres. capsomeres assemble into an icosohedral or irregular-shappedcapsid usually assume a helical form. some of the larger viruses have a lipid containing envelope that surrounds the capsid. in addition, many viruses have glycoprotein spikes that extend from the surface of the virus, acting as attachment projections or as enzymes.
specimen selection and collection:
specimen selection depends on the specific disease syndrome, viral etiologies suspected and time of year.
throat, nasopharyneal swab or aspirate: throat swabs are acceptable for recovering enteroviruses, adenoviruses and hsv, whereas nasopharyngeal swab oraspirate specimens are preferred for the detection of influenza and parainfluenza viruses. throat specimens are collected by rubbing inflamed, vesiculated, or purulent areas of the posterior pharynx with a dry, sterile swab. nasopharyngeal secretion specimens are collected by inserting a swab with flexible shaft through the nostril to the basopharynx.
rectal swabs and stool specimens: used to detect rotavirus, enteric adenoviruses and enteroviruses. rectal swabs are collected by inserting a swab (3-5) cm into the rectum to obtain feces.
urine: cmv, mumps, rubella, measles, polymaviruses and adenoviruses can be detected in urine. virus recovery may be increased by processing multiple (2-3) specimens because virus can be shed intermittently or in low numbers. the best specimen is at least (10 ml) of a clean-voided first-morning urine.
blood: used to detect cmv, hsv, enteroviruese and adenoviruses. (50-10 ml) of anticoagulated blood collected in a vacutaner tube is needed. heparinized, citrated or edta anticoagulated blood is acceptable for detection.
tissue: useful for detecting viruses that infect lung (cmv, influenza virus, adenovirus), brain (hsv), and gastrointestinal tract (cmv). specimens are collected during surgical procedures.
specimen transport and storage:
all specimens collected for detection of viruses should be processed by the laboratory immediately. specimens for viral isolation should not be allowed to sit at room or higher temperature. specimens should be placed in ice and transported to the laboratory at once. under unusual circumstances, specimens may need to be held for days before processing. for storage up to 5 days, hold specimen at 4°c. storage for 6 or more days should be at – 20°c or preferably at – 70°c.
viral transport media are used to transport small volumes of fluid specimens, small tissues and scraping, and swab specimens, especially when contamination with microbial flora is expected. examples of successful transport media include stuart’s medium, amie’s medium, hanks balanced salt solution and eagle tissue culture medium.
virus detection methods:
a. virus isolation:
1. embryonated eggs:
in order to cultivate viruses in eggs, the procedure adopted should be very simple. the eggs are kept in incubator and embryos of (7-12) days old are used. the egg containing embryo usually has an air apace at the larger end. the position of this sac is first determined. the shell over the air sac is then cut off and removed. the membrane adjacent to the shell is then pierced. usually the hypodermic syringe is used for piercing the shell. at this stage, the embryonic fluid may ooze out. the embryo then gets exposed and ready for use.
virus suspension to be cultivated is taken in dropingingingper and gently spread over the exposed embryo. after inoculation is thus completed, the open area of the shell is sealed eggs are incubated for one week as in hatching. the virus particles infect the membrane at random and create pock marked appearance against the transparent background. this indicate viral basis.
the embryonated offers several sites for the cultivation of viruses:
chorioallantoic membrane.
allantoic cavity.
amniotic cavity.
yolk sac.
2. laboratory animals:
mice are still most widely used animals in virology. mice can be inoculated through several routes, i.e. intracerebral, subcutaneous, intraperitoneal, intranasal. other animals such as rabbits and ferrets are also used. the growth of virus in inoculated animals is indicated by death, disease or visible lesions. animal inoculation has a disadvantage that immunity may interfere with viral growth and that animals often harbor latent viruses.
3. cell culture:
to detect virus using living cells, suitable host cells, cell culture media and techniques in cell culture maintenance are necessary.
host cells referred to as cell cultures originate as a few cells and grow into a monolayer on the sides of glass or plastic test tubes. cells are kept moist and supplied with nutrients by keeping them continuously immersed in cell culture medium.
cell cultures are incubated in a roller drum that holds cell culture test tubes tilted (5-7) degrees while they slowly revolve at 35 to 37°c. incubation of cell culture tubes in a stationary rack can be used in place of a roller drum. to counteract the ph decrease, a bicarbonate buffering system is used in the culture medium to keep the cells at physiologic ph (ph 7.2). phenol red a ph indicator that is red at physiologic, yellow at acidic and purple at alkaline once inoculated with specimen, cell cultures are incubated for (1-4) weeks depending on the viruses suspected. periodically the cells are inspected microscopically for the presence of virus, indicated by areas of dead or dying cells called cytopathic effect (cpe).
two kinds of media, growth medium and maintence medium, are used for cell culture. usual antimicrobials added are vancomycin (10 mg/ml), gentamicin (20 mg/ml) and amphotericin (2.5 mg/ml).
a cell culture becomes a cell line once it has been passed or subcultured in vitro. cell line are classified as:
primary cell lines, ex. primary monkey kidney cell.
low passage (semi-continuous) cell lines, ex. lung fibroblast.
continuous cell lines, ex. human epidermoid carcinoma cells.