Viral Identification Detection by CPE Detection Of Viral

Viral Identification- Detection by CPE.

Detection Of Viral Pathogens by Culture • Viruses are obligate intracellular parasites • Some viruses can be propagated or cultured in susceptible hosts – whole animals – mammalian cells grown in culture • Virus assays in cell cultures by quantal e. g. , enumerative methods (plaque or local lesion assays)

Virus Detection in Cell Culture • Some viruses (some enteroviruses, reoviruses, adenoviruses and astroviruses) propagate in susceptible host cell cultures and produce morphologically distinct cytopathogenic effects (CPE). Uninfected Cell Culture Infected Cell Culture with CPE

Virus Isolation / Cell culture (V 3. 6 b) Inoculate and wait. . . • for cytopathic effect (CPE). • Changes in the nature of the cells that occurs as a result of viral growth. • CPE = “a virus is growing” • False positives – Viral contamination, cell contamination, toxins, aged cells – Always include a non inoculated control.

Advantages and Disadvantage • • Isolation of the virus has one big advantage: allows to obtain high titres of infectious virus present in clinical specimen. Disadvantages: • requires long time • leads only to initial identification of isolated virus • some important viruses cannot be cultured • there is always risk of laboratory infection with cultured virus • experienced personnel is needed • expensive

CMV CPE

Inculcation of Viral specimen • • - 1 - Suspension Method. 2 - Monolayer method: Discharge the old medium. Add 500μl-1 ml from the sample. Incubation in 37°C for 30 -1 hour. Washing by PBS. Add new maintenance medium. Incubation at 37°C for suitable time, and fallow.

Challenges in Assaying Viral Infectivity • Some viruses (some enteroviruses, adenoviruses, rotaviruses, astroviruses and hepatitis A virus) grow poorly or slowly in cell cultures and produce little or no CPE. • Greater detection with additional analytical techniques: – Viral antigens • Immunofluorescence assays, enzyme immunoassays, radioimmunoassays, etc. – Viral nucleic acid assays: hybridization and/or amplification • Combined cell culture + RT-PCR demonstrates presence of greater numbers