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RSV pseudotypes. Usually, a 1:5 dilution of heat-inactivated This novel method allows for three very common subgroups of
(56°C for 30 minutes) serum is mixed with an equal quantity exogenous ALVs (ALV-A, ALV-B, and ALV-J) to be detected and
of a standard preparation of RSV of a known pseudotype. differentiated in one reaction. Real-time RT-PCR using the H5/
After incubation, the residual virus is quantitated by any one H7 primer pair has been shown to be highly reproducible (Kim et
of many procedures, the cell culture assay being most com- al., 2002; Qin et al., 2013). TaqMan-based real-time PCR method
monly used. A microneutralization test to assay for residual for the rapid detection and quantification of ALV-J with proviral
virus can be used for detection of ALV antibody (Fadly and DNA (Qin et al., 2013) as well as ALV-A and ALV-B (duplex
Witter, 1998). The test can be conducted in 96-well plates, real-time RT-PCR) (Zhou et al., 2011) have also been reported.
and the neutralization of the virus is determined by ELISA Dai et al. (2015) showed that the SYBR Green I-based real-time
on culture fluids (Fadly et al., 1989). Positive ELISA indicates RT-PCR assay provides a powerful tool for the detection of ALV
no antibody, whereas a negative ELISA indicates neutraliza- and study of virus replication and infection. In a recent report, Xie
tion of ALV and the presence of antibody. Although virus and colleague used a proximity ligation technique and combined
and antibody within subgroups usually cross-neutralize, PCR with the immunoassay to develop a novel immuno-PCR
antibodies against variant viruses may fail to neutralize (Im-PCR) approach for the detection of ALV (Xie et al., 2016).
a representative subgroup virus. This has been observed As further modification of PCR tests, loop-mediated isother-
particularly with subgroup J, due to virus mutation, and is a mal amplification (LAMP) method for ALV subgroup A and J
limitation to the use of VN tests. These tests are slow requir- has been developed (Zhang et al., 2010; Wang, Y. et al., 2011).
ing seven to ten days and are technically demanding. The LAMP method was developed by Notomi et al. (2000).
2 Antibody ELISA tests. Viral antigens may be used in ELISA This novel technique generally requires isothermal conditions
tests to detect subgroup-specific ALV antibodies. Tests for and four different primers for DNA amplification (Notomi et al.,
antibodies to subgroups A, B and J are commercially avail- 2000; Mori et al., 2001) and has been applied to the detection
able. The subgroup J antibody ELISA use recombinant env of several pathogens. The LAMP reaction requires 30 to 60 min
antigen produced in baculovirus system (Venugopal et al., and can be performed at a single temperature ranging from 60 to
1997) and appear to detect antibodies to all variant viruses 65°C. LAMP does not require the DNA denaturation, annealing,
studied. The antibody ELISA tests are rapid (requiring one and extension PCR cycles (Notomi et al., 2000; Mori et al., 2001).
day), specific, and suitable for large scale testing. In addition, the results can be ascertained easily by the naked eye
(Mori et al., 2001; Bista et al., 2007).
Molecular techniques
A more effective control of ALV infections mainly depends on the
early detection and removal of infected birds to reduce contact Intervention strategies
with non-infected birds and the incidence of horizontal spread.
Therefore, achieving rapid detection of infection is imperative in Vaccination
effective control of the spread of ALVs. Polymerase Chain Reac- Vaccination strategy is not preferred for the control of ALV infec-
tion (PCR) based methods had been developed to provide a rapid tions, as eradication of the virus from the flocks is preferred.
tool for detection and identification of ALVs proviral DNA and However, the idea of using vaccines to increase host resistance
viral RNA including subgroup E viruses. Reverse transcription to ALV infection has been proposed (Salter et al., 1991). In a
PCR (RT-PCR) has also been used to detect several subgroups series of attempts to inactivate ALV by various means, however,
of ALV (Häuptli et al., 1997; Zhou et al., 2011). A specific PCR Burmester (1968) demonstrated that ability of these virus
for ALV subgroup A can be used to detect proviral DNA and viral preparations to induce antibody was destroyed almost concur-
RNA in various tissues from ALV-infected chickens (van Woen- rently with inactivation (unpublished data). Attempts to produce
sel et al., 1992). Reverse transcriptase nested PCR (RT-nested attenuated strains of ALV that do not induce disease have also
PCR) test that amplifies a fragment of the LTR of exogenous failed (Okazaki et al., 1982). Results of experimental vaccina-
ALV subgroups A, B, C, D and J, but not endogenous retroviral tion with live ALV on shedding and congenital transmission
sequences, has been described (García et al., 2003). Several of the virus are equivocal. Some success has been obtained in
primers specific for the detection of the most commonly isolated attempts to increase the resistance of the host to RSV by immu-
ALVs, particularly subgroup A (Lupiani et al., 2000), and the new nization with viral or cellular antigens (Payne, 1981; Bennett and
subgroup ALV-J (Smith, E.J. et al., 1998; Smith, L.M. et al., 1998; Wright, 1987). The use of experimental recombinant ALSVs as
Silva et al., 2000) have been developed. Other primers specific vaccines may prove to be a valuable adjunct to current programs
for endogenous, subgroup E ALV can also be used to detect cell for reduction or eradication of ALV infection. Recombinant
culture infected with endogenous ALV-E, but not those infected ALV-J gp85 protein vaccine with either a liposomal, a cytosine-
with exogenous ALV of subgroups A, B, C, D, and J (Fadly and phosphate-guanine oligodeoxynucleotide (CpG-ODN) or silica
Witter, 1998). Multiplex PCR is a useful technique for the rapid nanoparticles adjuvant provided partial protection and elicited
differential diagnosis of avian viruses and the detection of multi- high antibody titres (Dou et al., 2013; Zhang et al., 2014; Zhang
ple infections of avian viruses under field conditions. Recently a et al., 2015; Cheng et al., 2017). Immune enhancer Taishan Pinus
sensitive and specific multiplex PCR method for the detection of massoniana pollen polysaccharide (TPPPS) reportedly can
ALV-A, ALV-B, and ALV-J has been developed (Gao et al., 2014). enhance the immunogenicity of gp85 recombinant proteins and