Saturday, August 13, 2011

Laboratory diagnosis of viral infections :Virus isolation

null It is still the gold standard method for virus detection. Traditionally, three methods are used for virus isolation; cell culture, animal inoculation and embryonated eggs. Animal inoculation is extremely costly and used only in research laboratories e.g. certain Coxsakie A viruses are isolated in suckling mice and embryonated eggs are rarely used e.g. Influenza viruses. Cell culture is the most commonly used by clinical virology laboratories (Nauschuetz and Learmonth, 2007).

Traditional cell culture
There are three basic types of cell cultures. Primary cultures are obtained from tissue removed from an animal. The tissue is finely minced and then treated with trypsin to disperse cells then the cells seeded onto a surface to form monolayer, as flask or test tube. Primary cell lines can only be passaged a few times e.g. primary monkey kidney cell (Nauschuetz and Learmonth, 2007).
Diploid cell lines are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages); with increasing passage diploid cells become more insensitive to viral infection e.g. human neonatal lung culture. Continous cell lines have variable number of chromosomes (haploid) and are capable of more prolonged perhaps indefinite growth that have been derived from diploid cell lines or from malignant tissues e.g. HEP2(derived from human laryngeal carcinoma). The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus (Knipe, 2001).

Table (3): shows Different cell lines sensitive to common viruses (Specter and Bendinelli, 2005).
Cell line Viruses
A549 cells Adenovirus, HSV.
HeLa cells HSV.
HEP-2 cells Adenovirus, HSV, RSV.
Human embryonic kidney Adenovirus, BK virus, HSV.
Human diploid fibroblasts CMV, HSV, VZV, Enteroviruses, Rhinoviruses.
Madin-Darby canine kidney (MDCK) Influenza viruses.
NCI-H 929 Adenovirus, Enteroviruses, Mumpes,
Primary monkey kidney (PMK) cells Measles virus, PIV, RSV.
Primary rabbit kidney cells Enteroviruses, Influenza viruses, PIV.

Centrifugation-Enhanced shell vial culture
This technique is simple method that more rapidly identifies the virus than the traditional viral culture. Cells are grown on a round coverslip in a shell vial. The shell vial is inoculated with the clinical sample and then centrifugated to promote viral absorption. The shell vial is incubated for 24-48 hours, after which the cells are scraped from the coverslip, and the DFA technique is performed using a varity of Abs (Nauschuetz, 2000).
Identification of viruses detected in cell culture:
Detection of virus in cell culture depend on cytopathic effects (CPE) which are morphological changes noted as a result of viral replication. CPE of cells may be clumping, destruction, granulation, rounding or vaculation, giant cell or syncytia formation, or retractile cells. In some circumstances, viruses will not cause a visible CPE and it is necessary to resort to other methods to detect their presence. Mumps, Influenza and Parainfluenza viruses will not normally cause CPE or little CPE but addition of guinea pig red blood cells (RBCs) to infected primary monkey kidney cells will result in adherence of the RBC to the cells (hemadsorption) and Rubella virus may be detected by interference with echovirus 11 to replicate and cause cell destruction of African green monkey kidney cells (Smith and Yassin, 2000).

No comments: