What is the most reliable test for avian retrovirus identification and isolation?
Virus isolation/detection
A variety of samples may be collected from a flock for detection of the presence of, or for isolation of, ALV or other tumor viruses. The presence of infection can be demonstrated most easily by detection of antibodies in serum. Viruses can be detected in and isolated from serum, plasma, buff y coat cells, tumor tissue, normal parenchymatous tissues (e.g. liver), feather pulp and tips, meconium, vaginal and cloacal swabs, egg albumen and embryos. Because the viruses are thermo labile at room temperature, samples should be collected from live or freshly killed birds, or from newly laid eggs, and stored and shipped at _70_C.
A variety of diagnostic tests are available for the detection of ALSV. The principles of the commonly used tests are outlined here; detailed procedures are available in laboratory manuals and technical article.
Virus isolation is generally the ideal detection method, since the technique can detect all ALVs and is the starting point for several other investigative methods. Material (normally cell-free) to be tested for exogenous ALV is inoculated into specific-pathogen-free C/E chick embryo fi broblasts (CEFs) (susceptible to all ALV subgroups except E) growing in tissue culture plates or wells of microtitre plates, and the cultures are incubated for 7 days. Supernatants are collected and stored at _70_C as virus isolates for other studies. The CEFs are sonicated and tested for the presence of ALV p27 gs antigen in an antigen enzyme-linked immunosorbent assay (ELISA) test (commercially available). A positive optical density (OD) reading for gs antigen compared with uninoculated CEF cultures (in which low levels of gs antigen of endogenous ALV origin may be detected) is indicative of the presence of exogenous ALV of subgroups A, B, C, D or J. Ideally, C/E CEFs from chickens free of endogenous gs antigen, such as line 0, should be used. If necessary, subgroup E ALV can be identified by inoculating test material on C/E CEF and quail embryo fi broblasts of phenotype Q/BJ. Subgroup E ALV will grow in the Q/BJ cultures but be excluded from the C/E cultures.
An antigen ELISA is available commercially for the detection of ALV p27 gs antigen in various materials such as egg albumen, meconium, vaginal and cloacal swabs, and feather pulp, as evidence of ALV infection. The test is used particularly to detect ALV shedder hens in ALV eradication programmes and is cheap, rapid and suitable for large-scale use. It is not subgroup specific for the different exogenous ALV subgroups (which is usually of no concern) but it has the disadvantage of also detecting gs antigen of endogenous ALV origin, although usually at
a lower level. The testing of serum or plasma for gs antigen is not recommended because of the confusion between antigen of endogenous and exogenous origin. The antigen ELISA is also used to detect ALV growth in virus isolation tests in CEF as discussed above.
Virus characterization
The envelope subgroup of ALV isolates may be determined by several methods:
• Viral interference assays, which test the ability of an ALV isolate to prevent focus formation in C/E CEF cultures by RSV strains of known subgroup.
• Virus neutralization assays, in which the isolate, or the RSV pseudo type of the isolate, is exposed to antisera with known neutralization specify city to each subgroup, and examined for growth, or focus formation, respectively, in C/E CEF cultures (N.B. An RSV pseudo type is an RSV that can be created by coating a replication defective RSV with the viral envelope of an ALV, endowing the pseudo type with the subgroup characteristics of the ALV)
• Host range assays, in which the isolate, or the RSV pseudo type of the isolate, is placed in a subgroup depending on its ability to grow in, or transform, CEF cultures of varying ALV subgroup susceptibility phenotypes
• Polymerase chain reaction (PCR) assays. Primer pairs are available specifi c for the diff erent ALV subgroups, including J. A problem with PCR methods is that the primer pairs used may not detect all variant viruses. Reverse-transcriptase PCR has also been developed for detection of viral RNA. PCR-induced sequences of various retroviral genes can be used to study viral relatedness and variability.
Serology
Detection of antibodies is used in flock surveillance to determine the presence or absence of infection by ALV of different subgroups, and to help to characterize the infective status of individual birds in epidemiological and eradication studies. Two types of test are mainly used:
• Virus neutralization tests, in which antibody is detected by ability to neutralize infectivity of ALVs, or RSV pseudotypes, of known subgroup; these tests are technically demanding and time-consuming
• Antibody ELISA tests. Tests for antibodies to subgroups A, B and J are available commercially and are rapid and suitable for large-scale testing.
A variety of other tests are available for testing for ALV and ALV antibodies. These include: the no producer (NP) cell activation tests for ALV; the phenotypic mixing test for ALV; the complement fixation test for ALV (COFAL); fluorescent antibody and other immunohistochemical tests for ALV and antibody; in situ hybridization for ALV; and chick and embryo inoculation tests for ALV pathogenicity. Highly sensitive tests for reverse transcriptase have been used to screen human vaccines produced in CEF for freedom from avian retroviruses. Details of these various tests can be found in laboratory manuals.
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