Respiratory Viruses Slide Set

Laboratory Diagnosis of Adenoviruses Infection

D. Laboratory Diagnosis

Virus Isolation - Adenovirus may be isolated from most bodily fluids and secretions; from eye swabs, NPA, Throat swabs, urine, faeces, leucocytes and the CSF. The most reliable source for isolation is faeces, particularly for members of subgenera A, D, and F. Members of subgenera B, C, and E can also be isolated form the urine. Adenoviruses are stable and do not need refrigeration during transport. HEK cells are permissive for growth of all adenovirus strains except the enteric strains Ad 40 and Ad41. Hep-2 cells can also be used. Primary monkey kidney cells and 293 cells will permit the growth and isolation of Ad 40 and Ad41 strains. Adenoviruses produce a characteristic CPE in cell culture, which consist of the rounding and clustering of cells with refractile intranuclear inclusion bodies. The CPE may start at the periphery of the culture before generalizing. However, some of the group B adenoviruses do not cause cell enlargement or clustering.

The isolate can be positively identified as an adenovirus by complement fluorescent antibody, complement fixation or ELISA tests, using antibodies against the common group antigen (EM may also be used). The adenovirus can be typed by the use of neutralization tests or HAI tests. (NB. The adenovirus CF antigen is a property of the hexon that contains group specific as well as type specific Ag. The HA antigen is carried by the pentons which has type-specific antigenic epitopes. Neutralization properties are intrinsic to both the external part of the hexon and the fiber) The FA test using a hyperimmune serum to any of the common adenovirus serotypes will react with the rest of the human adenoviruses. Recently, type-specific monoclonal antibodies have become available for use in fluorescent antibody, ELISA, and LA tests for differentiating enteric Ad 40 and Ad 41 from other adenovirus strains. The isolate can also be typed by hybridization and restriction endonuclease digestion patterns.

Detection of Ag by IF - Direct microscopic examination of exfoliated cells is not generally successful. However, with the use of IF techniques against the common adenovirus antigen, a sensitivity rate approaching that of routine tissue techniques have been reported. However, some researchers using IF have detected only one-third of the NPA from which adenoviruses were subsequently isolated in tissue culture. Furthermore, IF has not been useful in the early diagnosis of conjunctival adenovirus lesions but may be useful in diagnosing acute haemorrhagic cystitis.

Serology - Mammalian adenoviruses share group-specific antigen epitopes (on the inside of hexons) which is recognized by complement fixing antibodies. Complement fixation for adenovirus- specific antibodies is ideally performed with a pool of antigen representing a serotype of each subgenus. If this is hard to accomplish, representative serotypes of subgenus B and C should be used. The extract should be prepared from sonicated infected cells which will contain a 10 fold excess of free hexons. ELISAs have also been prepared against the common group antigen. Because many individuals would have experienced recent infection with another adenovirus, and thus already have a high titre of AB against the group Ag, it was suggested that CFTs will only detect 50% of new infections. To type the virus, HAI and neutralization tests would have to be used. For HAI tests, rat erythrocytes should be used for subgenus A, C, D, E or F, and monkey erythrocytes for subgenera B. Serum neutralization assays are cumbersome but are the most efficient means of detecting specific antibodies against each adenovirus serotype.  

E. Treatment and Prevention

There is no specific antiviral chemotherapy against adenoviruses at present. Idoxuridine and ARA-A had been tried in the treatment of keratoconjunctivitis but were unsuccessful. Antivirals have not been tried for the adenovirus-induced respiratory syndromes. When these diseases occur, they are difficult to distinguish from similar diseases caused by a variety of RNA viruses. Swimming pool-associated conjunctivitis can be prevented with adequate levels of chlorine in the water.

Effective vaccines are available for use in the military against adult respiratory distress syndrome. These vaccines are not currently licensed for administration to civilians. Non- attenuated live Adenovirus types 4, 7, and occasionally 21 are packaged into enteric capsules so that they bypass the respiratory epithelium and only replicate once they reach the intestine, resulting in an asymptomatic infection of the intestine. These strains were cultivated in human embryo fibroblasts. Earlier adenovirus vaccines were grown in primary monkey cells that have been demonstrated to contain SV40 virus. SV40 virus genome can integrate into the E3 region of adenovirus, indeed a recombinant adenovirus was isolated which contained the tumourigenic T antigen of SV40. As a result, this approach to producing a vaccine was abandoned. At least 15 million doses of adenovirus vaccine had been given and the vaccine had been shown to be both safe and effective. Adenovirus-associated pneumonia has ceased to be a major problem in the US army. The vaccine has not been licensed for use in children because of worries over the lack of attenuation and the potential oncogenic potential of these vaccines.  

Respiratory Viruses Slide Set