Page 75 - Avian Virology: Current Research and Future Trends

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Therefore, when PPMV-1 is suspected, it is advisable to use both tested by the F gene RRT-PCR assay. The F-gene based assay was
cell culture and ECE. developed specifically to detect virulent NDV from field swabs
during the 2002 ND outbreak in the USA (Wise et al., 2004b).
Although the assay can identify a wide range of isolates from
Virus characterization different parts of the world, it fails to identify some PPMV-1 iso-
The HI test is commonly used to identify NDV. In addition, lates (Kim et al., 2008). These results suggest that because NDV
panels of monoclonal antibodies can be used to characterize isolates show considerable variation in the nt sequence around
and group isolates (Alexander et al., 1987; Lana et al., 1988). the F protein cleavage site, the designed F gene probe may not
Although monoclonal antibodies are very helpful in diagnosis recognize all NDV isolates.
of NDV, their ability to detect a broad range of NDV isolates is Both OIE and USDA define an APMV-1 isolate virulent (noti-
often limited. Other serological tests, such as ELISA (Snyder et fiable disease), if the isolate has an ICPI value equal to or greater
al., 1984; Adair et al., 1989), virus neutralization assay in chicken than 0.7 and/or presence of at least three basic aa (arginine or
embryos (Beard, 1980) and plaque neutralization test (Beard and lysine) between residue positions 113 and 116 of the C-terminus
Hanson, 1984) are available, but they have limited value in assess- of the F2 protein and a phenylalanine at position 117, which is the
ing the pathogenicity of the isolate. Historically, three in vivo tests N-terminus of the F1 protein.
have been used to determine the pathogenicity of NDV isolates:
(1) mean death time (MDT) in ECE, (2) ICPI test and (3)
intravenous pathogenicity index (IVPI) test (Alexander, 2009). Immune response to NDV
The MDT is the mean time in hours for the minimum lethal The immune response to NDV varies depending on the infecting
dose to kill chicken embryos. The MDT has been used to clas- strain and the host species. NDV infections caused by lentogenic
sify NDV isolates as velogenic (taking less than 60 hours to kill), or mesogenic strains are usually resolved by host innate and adap-
mesogenic (taking between 60 to 90 hours to kill), and lentogenic tive immunity. But in the case of velogenic strains, the virus might
(taking more than 90 hours to kill). The ICPI test is performed by simply outrace host defence and quickly overwhelm the host
inoculating 0.05 ml of a 1:10 dilution of fresh infectious allantoic leading to death. NDV is a highly infectious and a fast-replicative
fluid into the brain of each of 10 1-day-old chicks. The birds are virus. Therefore, both strong innate and strong adaptive immune
observed daily for 8 days. At each observation, a bird is scored 0 responses are needed for complete protection against NDV infec-
if normal, 1 if sick and 2 if dead. The ICPI value is the mean score tion.
per bird per observation. The velogenic viruses give indices that The host innate immune response is the immediate reaction
approach the maximum score of 2.0; whereas, lentogenic viruses to NDV infection, which is aimed at resisting virus growth and
give values close to 0.0. IVPI is a weighted score of clinical signs aiding the host to develop specific protection from the adaptive
after intravenous injection of the virus into 10 six-week-old chick- immune response. The innate immunity is critical in the early
ens. Lentogenic and some mesogenic strains have IVPI values period of infection. NDV enters the host through the respiratory
of 0.0; whereas, the indices for velogenic viruses approach the tract where it first encounters extracellular innate defence barriers,
maximum score of 3.0. Of the three tests, ICPI is internationally which can inactivate the virus particles and signal for recruitment
used for determining the virulence of NDV isolates, because of its of immune cells. Among various extracellular defence barriers,
sensitivity and reliability (Alexander, 1988). However, the ICPI complement (C′) is a powerful system of host early innate immune
test may not reflect the true pathogenicity of viruses isolated from response. It has been shown that NDV can be inactivated by C′ in
species other than chickens. For example, some PPMV-1 isolates the absence of antiviral antibody (Welsh, 1977). It has also been
have ICPI values of mesogenic strains but have MDT values of shown that NDV can activate all three C′ pathways (Biswas et al.,
lentogenic strains (Pearson et al., 1987). Therefore, the patho- 2012). NDV was found to passively incorporate host cell regula-
genicity of a non-chicken NDV isolate for chickens should be tors of complement activation (RCA) proteins CD46 and CD55
determined by experimental infection of chickens using natural at levels that could provide virus protection against C′-mediated
routes of infection. neutralization. It was also shown that NDV grown in ECE were
The conventional pathogenicity tests are time consuming not neutralized by non-immune chicken serum but were neutral-
and expensive; hence, several rapid pathotyping methods such ized by normal human serum, confirming that RCAs function in
as reverse transcription (RT)-PCR and sequencing of the F a species-specific manner (Biswas et al., 2012).
protein cleavage site have been developed (Miller et al., 2010). Studies have shown that pro-inflammatory cytokines,
Of these assays, the real-time RT-PCR (RRT-PCR) offers the chemokines, type I and type II IFNs, and antiviral proteins deter-
highest sensitivity. Several RRT-PCR assays have been developed mine the early host innate immune response to NDV infection
in the last decade around the world to detect viruses circulating (Ecco et al., 2011; Rue et al., 2011). For example, IL-6, MDA-5,
in their locations (Miller et al., 2010). In the USA, two different and IFIT-5 in the spleen of infected chickens have been shown to
RRT-PCR assays have been extensively used (Wise et al., 2004b). be induced by a virulent NDV strain at 1-day post infection (Rue
An M-gene based assay was developed for the detection of NDV et al., 2011). Further studies have shown that chickens infected
isolates (Kim et al., 2007a,b). However, due to sequence variation with different velogenic genotypes induced different viral load
of the M gene, some NDV isolates are not detected by this assay. and different levels of cytokines and chemokines associated with
Therefore, samples testing positive by the M gene are subsequently inflammatory reactions (Hu et al., 2012; Rasoli et al., 2014). NDV