To determine an appropriate NDV PCR to use for the simultaneous detection and differentiation of virulent and avirulent NDV strains A desktop study was undertaken to determine the best PCR available for the simultaneous detection and differentiation of virulent and avirulent Avian Paramyxovirus 1 (APMV-1) strains. The most appropriate PCR for the differentiation of virulent from avirulent APMV-1 strains was determined to be the real-time reverse-transcriptase PCR developed by Fuller et al. (2009). This PCR employs two probes; one to detect virulent APMV-1 (probe VRP2) and one to detect avirulent APMV-1 (Probe ARP2). The test is designed to work for Class II NDV’s (such as with the Wise PCR test) but not Class I NDVs (such as those samples positive in the Kim PCR test).
The probes are designed to cover the cleavage site sequence of the F-gene. They incorporate inosine in positions where the nucleotide ambiguity is greatest and locked nucleic acids (LNAs) at nucleotide positions that encode the amino acids which are determinate for virulence. LNAs increase the thermal stability of probe/template duplexes and improve selectivity compared to the corresponding unmodified reference strands. Thus LNAs act to improve the ability of the probe to hybridise with sequence variants that have multiple mismatches between the probe and the target. All further work was conducted with this test, and is referred to in this report as “virulent/avirulent multiplex qPCR”.
To confirm the ability of the new virulent/avirulent multiplex qPCR to correctly identify a virus as virulent or avirulent, a range of known virulent and avirulent samples were tested including recently obtained virulent APMV-1 strain from pigeons, a sample containing the avirulent vaccine strain (V4) and a range of reference organisms supplied by the national NDV reference lab (CSIRO AAHL). All of the avirulent samples were correctly identified using this PCR (Refer to Table 1). However, one known Class II virulent strain of NDV (sample 20 in Table 1) that was positive in the Wise PCR test, was not able to be detected in the virulent/avirulent multiplex qPCR (highlighted in green in Table 1). It is not known why this sample was not detected in the virulent/avirulent multiplex qPCR given that it gave a strong positive PCR result in the Wise PCR test. One explanation may be that the degree of mismatch in the probe was more than able to be tolerated in the PCR and therefore the probe was unable to bind to the target DNA. One other sample that did not give a clear result in the virulent/avirulent multiplex qPCR was sample 11 (a mesogenic chicken sample from Komarov). This sample gave a clear strong positive result in the Wise PCR and then gave a clear and strong result with both virulent and avirulent probes in the multiplex qPCR, rather than with just one or the other probe. This may have something to do with the fact that this sample is classified as mesogenic and may contain sequence similarities with both the velogenic and lentogenic probes.
Despite the fact that two samples that did not give clear results, the virulent/avirulent multiplex qPCR appears to be an appropriate choice of test to use to determine whether a Class II APMV-1 sample is virulent or avirulent.
Table 1: Comparison of results for the virulent/avirulent APMV-1 qPCR and the Wise and Kim qPCR tests for APMV-1. Green shading indicates where the virulent/avirulent PCR test did not give the expected result.
# Confirmed as being virulent using a specific PCR for the APMV-1 strain infecting pigeon in Victoria in 2012 at the CSIRO AAHL laboratories (J. Wang Pers. Comm.)
* Ct refers to the crossing threshold value in Real time PCR. Typically a value >40 is negative, and Ct values < 36-40 are positive (this value can vary depending on the test).
a Sample tested negative in Kim/Wise PCR and so was repeated on CSIRO AAHL NDV PCR
NT Not tested
To screen a selection of NDV positive and negative wild bird samples previously tested in Kim/Wise PCR using the new PCR to compare diagnostic sensitivity A total of 62 diagnostic and wild bird samples of known APMV-1 status were screened in the new virulent/avirulent multiplex qPCR, and the Wise and Kim qPCR tests (the Kim/Wise multiplex qPCR was evaluated by BSL (DAFF) and co-workers as part of a previous WEDPP project funded in 2010/2011). The samples were selected to represent a range of positive and negative APMV-1 samples and included samples from ducks (10), chickens (49) and pigeons (3)(Refer to Table 2).
All samples that produced a negative result (Ct value of >40) in the Kim/Wise multiplex qPCR test also produced a negative result (Ct value of >40) in the virulent/avirulent multiplex qPCR test. However, 12 samples that produced a positive result in the Wise qPCR test did not produce a positive test result (Ct value of <40) in the virulent/avirulent multiplex qPCR test (For example Table 2; sample numbers 1, 2, 4, 5 , 7, 8 and 10; highlighted in green). In other words, the new PCR gave some false negative results. In all cases where the new virulent/avirulent multiplex qPCR failed to detect the APV-1 genome, the Ct value in the Wise test was greater than 27-28 (weaker positive result). These results suggest that the new virulent/avirulent multiplex qPCR is not as sensitive as the Kim/Wise qPCR and consequently this test would not be recommended as the screening test, but instead be used as a follow up test on Wise qPCR positives.
Ct values for samples which produced positive results for both tests were, in general, 1.5-3 Ct values higher in the virulent/avirulent multiplex qPCR (For example Table 2; sample numbers 15-22) than in the Wise qPCR test. This again supports the fact that the virluent/avirulent multiplex qPCR is not as sensitive as the Kim/Wise multiplex qPCR.
To further test the diagnostic sensitivity of the new virulent/avirulent multiplex qPCR, an additional 460 wild bird samples were screened for the presence of APMV-1 using the Kim/Wise qPCR (refer to Table 3). All wild bird samples were from ducks and included the following species: Pacific black duck, grey teal and unidentified duck species. All samples originated from cloacal samples collected from individual birds. Of the 460 samples screened in the Kim/Wise qPCR, a total of 31 were positive for APMV-1, and 429 tested negative for APMV-1. Surprisingly, all 31 APMV-1 Wise PCR positives tested negative in the new virulent/avirulent multiplex qPCR. This was repeated several times to check for error, however the result remained the same at each testing. The most likely explanation is that the samples were below the limit of detection for the virulent/avirulent multiplex qPCR given that all samples produced a Ct value above 28.60 in the Wise PCR. When stronger positives are encountered in the Wise PCR, the virulent/avirulent multiplex qPCR appears to give more reliable results. Based on these results, we would not recommend the new virulent/avirulent multiplex qPCR for wild bird samples with a low positive result (eg Ct >28) in the Wise PCR.
It was decided to try and further improve the diagnostic sensitivity of the virulent/avirulent multiplex qPCR, given that testing of samples with a high Ct using the Wise/Kim multiplex qPCR yielded negative results using the virulent/avirulent multiplex qPCR. All class II APMV-1 samples with a Ct value of approx >28 using the Wise/Kim multiplex PCR gave no amplification (Ct >40) in the virulent/avirulent qPCR. To improve the sensitivity of the virulent/avirulent qPCR, the concept of a nested PCR was introduced. The first round of the nested PCR used the F-gene primers described by (Collins et al., 1993) which were previously used to characterise Australian NDV isolates (Peroulis-Kourtis et al., 2002; Peroulis & O'Riley, 2004). For the second round of the PCR, the same virulent/avirulent multiplex qPCR as described in this report (Fuller et al, 2009) was employed. As a proof of concept, the nested qPCR protocol was tested on strong and weak positive avirulent control samples as well as the LEADDR network quality control sample which is derived from a virulent NDV strain (Table 4).
The Ct values for nested virulent/avirulent APMV-1 qPCR on the strong and weak positive avirulent samples improved markedly from those obtained with just the standard virulent/avirulent multiplex qPCR (Table 4). The LEADDR network quality control samples that tested negative in the standard virulent/avirulent APMV-1 qPCR test, tested positive in the nested virulent/avirulent APMV-1 qPCR test although the Ct values for the three replicates varied (Table 4). Further testing of this protocol is required to confirm that it is able to consistently produce amplification products from samples which produce Ct values >28 using the Wise/Kim multiplex PCR. It is also suggested that further work needs to be done to identify if the primer pair used for the first round PCR in the nested virulent/avirulent APMV-1 qPCR are able to amplify the F-gene of all known variants of the virus circulating at present.
Table 2: Virulent/avirulent APMV-1 qPCR and Wise/Kim qPCR results for a range of bird samples. Yellow shading indicates where the different PCR test results correlate; green shading highlights where the virulent/avirulent PCR did not give the expected result.