Page 245 - Avian Virology: Current Research and Future Trends

238 | Yao and Nair

and other breeder flocks for freedom from virus infection. Virus detection of oncogenic RNA viruses including all ALSVs (Temin,
isolation is generally considered the ‘gold standard’ of ALV 1974). Detection of this enzyme, either directly using a correct
diagnosis. Samples most commonly used for detection of ALV template for detecting enzymatic activity (Kelloff et al., 1972;
include blood, plasma, serum, meconium, cloacal and vaginal Tereba and Murti, 1977) or indirectly using radioimmunoassay
swabs, oral washings, egg albumen, embryos and tumours (Fadly (Panet et al., 1975), is an indication of presence of virus.
and Witter, 1998; Fadly, 2000; Nair and Fadly, 2013). Virus also
can be isolated from albumen of newly laid eggs or the 10-day-old Characterisation of ALV subgroups
embryo of eggs laid by hens that are transmitting virus vertically, ALV isolates belonging to different subgroups can be character-
from feather pulp, and from semen. ALSVs are very thermolabile ized using following methods:
and long-term viability can be guaranteed only at temperatures
below –60°C. Thus, materials used for biological assays of infec- a Viral interference assays, which test the ability of an isolate to
tious virus should be collected and stored at –70°C until assayed. interfere with the formation of transformed foci in C/E CEF
Because most strains of ALV produce no visible morphologic cultures by RSV of known subgroup.
changes in cell culture, assays for the presence of ALV in infected b Virus neutralization assays, in which an isolate is placed in
cells are based on detection of specific viral proteins, or specific a subgroup according to susceptibility to neutralization by
proviral DNA or viral RNA sequences of ALV by the polymer- chicken antisera with known subgroup neutralizing activity.
ase chain reaction (PCR) and reverse transcription (RT)-PCR, The isolate, or the RSV pseudotypes, is exposed to anti-
respectively. Detection of ALV-encoded p27 forms the basis of serum and then examined for growth, or focus formation,
several diagnostic tests for virus. Indirect biologic assays such as respectively, in C/E CEF cultures. An RSV pseudotype is an
complement fixation for avian leukosis (COFAL), ELISA for ALV, acutely transforming RSV which can be created by coating a
phenotypic mixing (PM), resistance-inducing factor (RIF), and replication defective RSV with the viral envelope of an ALV,
non-producer cell activation (NP) tests are used for the detection and hence endowing the virus with the subgroup charac-
of ALVs. Of all these assays, ELISA is the most commonly used teristics of the ALV, in this case of the isolate. Fluorescent
test currently. Interpretation of the p27-based tests has to be done antibody staining of infected cultures using antisera against
with caution, as this antigen is shared by both exogenous and different subgroups is also used. These methods are usually
endogenous viruses and cannot differentiate between these two satisfactory, although in the case of subgroup J isolates, in
groups of viruses. All the virus isolation-based biological assays which env gene mutations and consequent antigenic changes
require the use of chicken embryo fibroblasts (CEFs) with specific are frequent, subgroup J antisera may fail to neutralize new
host range. CEFs that are resistant to infection with endogenous variants. However, mouse monoclonal antibodies have been
ALV (C/E) are desirable to use in tests for detection and isola- developed which appear in fluorescent antibody tests to
tion of exogenous ALV. Other cells, such as those resistant to detect a wider range of subgroup J isolates.
subgroup A (C/A) and resistant to subgroup J ALV (C/J) (Hunt c Host range assays, in which an isolate, or the RSV pseudo-
et al., 1999), can also be used to confirm the subgroup of isolated type, is placed in a subgroup according to ability to grow in,
ALV. Testing samples on CEFs that are susceptible to all ALV sub- or transform, respectively, CEFs of varying ALV subgroup
groups (C/O), and those that are resistant to subgroup E (C/E) susceptibility phenotypes. For example, a subgroup A ALV
can be used in differentiating exogenous and endogenous ALV. If would be identified if the virus grew in C/E CEFs but did not
a positive test is obtained from using C/O but not C/E CEFs, the grow in C/AE CEFs (resistant to subgroups A and E). This
sample is positive for endogenous ALV. Positive tests using both method relies on the availability of CEF phenotypes that
C/E and C/O indicates the presence of exogenous ALV. A flow exclude certain ALV subgroups. As such chicken lines that
cytometry method using a highly specific alloantibody termed R2 exclude subgroup J are not available, the use of a C/J CEF
has been described for detection of endogenous ALV envelope line that stably expresses J env gene has been suggested (17).
in chicken plasma (Bacon et al., 1996; Bacon, 2000). It should d Polymerase chain reaction assays specific for various ALV
be noted that some tests such as CF and ELISA and possibly subgroups have been developed (15, 36, 37), as discussed
non-producer NP, PM, R(-)Q cell, and FA can be suitable for later.
all leukosis and sarcoma viruses. The resistance-inducing factor
(RIF) test can be performed only on ALVs that are not rapidly Serological assays
cytopathogenic. Other tests are specific for certain virus strains. Detection of antibodies against ALV is used in flock surveillance
Rapid transformation of fibroblast cultures is produced only by to detect presence or absence of infection by exogenous ALV, and
certain RSV and of haematopoietic cell cultures only by defective to identify particular classes of birds in epizootiological studies
ALV. Direct (Kelloff and Vogt, 1966) and indirect (Payne et al., and ALV eradication programs. Plasma, serum, and egg yolk are
1966) immunofluorescence assay (IFA) as well as flow cytometry suitable samples for the detection of antibodies to ALSVs. Virus
(Hunt et al., 1999, 2000) have been used to detect viral antigen in neutralization and antibody ELISA can be used as described
CEF cultures. Flow cytometry has also been shown to be a very below:
useful tool in identifying ALV strains contaminating commercial
Marek’s disease vaccines (Fadly, 2006; Silva et al., 2007; Barbosa 1 Virus neutralization tests. Antibody is detected by its ability to
et al., 2008). Assays for RT activities have been used for the neutralize infectivity of ALVs of known subgroup, or of the