Discussion
NDV continues to cause outbreaks and substantial losses among the
poultry industry worldwide (Brown &
Bevins, 2017; Dimitrov et al., 2016). In
Egypt, in spite of the routine implementation of ND vaccination,
detection of NDV of genotypes 2.II, 2.VI, and 2.VII.1.1 have been
reported continuously over the last 10 years
(Orabi et al., 2017;
Saad et al., 2017;
Sabra et al., 2017). Continuously growing
phylogenic diversity points to the evolution of virus populations that
display a genetic distance to vaccine type NDV genotype 2.II. This
observation has been taken as indication for antigenic vaccine mismatch
and a suspected cause for vaccine failure
(Dimitrov et al., 2017;
Nagy et al., 2020). In this model new NDV
strains emerge because of an change in antigenic sites relevant for
protection. To test this hypothesis an “old” NDV isolate from 2011
representing NDV genotype 2.XXI was compared to a currently dominating
NDV genotype in chickens in Egypt (2.VII.1.), with specific focus on
sites blocking receptor binding and sensitive for neutralization.
Based on the new classification system proposed by
Dimitrov et al. (2019), viruses sequenced
in the current study were grouped phylogenetically as genotype 2.II,
2.XXI.1.1 and 2.VII.1.1. The latter originated from China and circulated
extensively in the Middle East (Ewies et
al., 2017; Radwan et al., 2013;
Saad et al., 2017). In contrast NDV
genotype 2.XXI has not been recorded in poultry for almost the last
decade. This group of viruses are descendent of genotype 2.XX and
together with the new genotype 2.VI form a monophyletic branch
(Dimitrov et al., 2019)(Dimitrov et al.
2019). Evolutionary time scale analysis indicate that genotype 2.XX
appeared around in 1959 in poultry (Chong
et al., 2013) forming sub-lineage 2.VIai that arose in Eastern Asia and
later circulated in Europe, causing sporadic ND outbreaks in Western
Europe and Bulgaria in the mid 1990s
(Ujvári et al., 2003) (Czegledi et al.
2002, Ujvari et al. 2003). In the second subgroup 2.VIaii
(Chong et al., 2013) that is now
consolidated in the new genotype 2.XXI, repeated chicken-to-pigeon
transmissions were observed that eventually was successful to establish
pandemic PPMV-1 infection, and forming the new genotype 2.VI (former
genotype 2.VIb) (Chong et al., 2013;
Ujvári et al., 2003). In addition, other
descendants of genotype 2.XXI established infections in the pigeon
population. These pigeon derived viruses are accumulated in three
sub-genotypes, i.e. 2.XXI.1.1; 2.XXI.1.2 and 2.XXI.2 respectively with
genotype 2.XXI.1.1 subsequently detected in Egypt
(Sabra et al. (2017). These phylogenetic
data highlight the possible interspecies transmission of genotype 2.XXI
viruses but clearly support the notion that isolates R1954/11-cl 1
R1973/11-cl 2 are original poultry derived strains and not a spill-over
infection from pigeon to poultry as described for PPMV-1 in the 1980s in
England (Alexander et al., 1985). This
notion of genotype 2.XXI being a virus circulating in poultry is further
supported by the finding, that closely related viruses were detected in
chickens in Ethiopia in 2011 and 2012 (De
Almeida et al., 2013). However, subsequent outbreaks in Egypt are
dominated by genotype 2.VII.1.1 viruses, indicating that genotype 2.XXI
was superseded. It was considered that ND vaccination is a driving force
for virus evolution (Chong et al., 2010)
with selection of escape variants (Cho et al., 2007; Cho et al., 2008).
In this respect genotype 2.VII.1.1. virus (AR 2178/2014) should harbor
mutations within neutralizing sites not present in genotype 2.XXI virus
(R1973/11-cl 2). However, when analyzing sites that are part of
neutralizing epitopes, all 6 mutations present in genotype 2.VII.1.1.
virus were also present in genotype 2.XXI virus. It was remarkable, that
for 5 out 6 sites, mutations were associated with a change of principle
properties of the amino acid, i.e. change from hydrophobic to alkaline,
positively charged (Y203H), from acidic, negatively charged to alkaline,
positive charged (N263K, E347K), from neutral to acidic, negatively
charged (G494D) or from hydrophobic to acidic, negatively charged
(V495E). That indicates, that mutations might be associated with a
change of antigenic properties. In line with this assumption are the
data from the cross HI-tests. Reactivity profile with mAb revealed
changes in specific epitopes and also polyclonal sera discriminated
between viruses from different genotypes. However, overall our data
support the notion that NDV / avian orthoavulavirus -1 is still a single
serogroup (Miller & Koch, 2013) and a
discrimination between genotype 2.XX and 2.VII was not evident. Taking
together our data do not indicate that a switch of antigenic sites were
the driving selection criterion for the replacement of genotype 2.XXI by
genotype 2.VII.1.1. This would be in line with the observation that the
overall mutation rate of NDV indicates strong purifying (negative)
selection for all proteins (Chong et al.,
2013; Miller et al., 2009).
It was striking that both historical 2.XXI isolates contained also a
vaccine type virus (genotype 2.II). Co-infections were revealed
initially by RT-qPCR but would have been concealed by regular
pathotyping using Sanger-sequencing: The F protein cleavage site of
virulent strains carries a polybasic motive
( 112R/K-R-Q-K/R-R*F117) and
avirulent NDV strains
harbor 112G/E-K/R-Q-G/E-R*L116 and a
leucine at position 116 at the F protein cleavage
site (Collins et al., 1993;
de Leeuw et al., 2005;
Peeters et al., 1999;
Römer-Oberdörfer et al., 1999).
For isolate R1954/11 the obtained polybasic cleavage site corresponded
to the ICPI, however pointing to a mesogenic pathotype. Sanger-sequence
of isolate R1973/11 pointed to avirulent pathotype, but the ICPI result
clearly revealed a velogenic pathotype. Such apparently contradicting
results have been published before (Nagy
et al., 2020; Tan et al., 2008), but
were not further resolved. After observing such contradicting results,
we plaque-cloned both isolates and were able to separate two different
NDV: beside genotype 2.XXI virus, a vaccine type virus (genotype 2.II)
was obtained. Subsequent pathotyping of the cloned viruses by ICPI now
matched with the sequence of the proteolytic cleavage site and full
genome sequence comparison of the sequenced lentogenic strains reveal a
close relation to vaccine strain Hitchner B1 / JN872151 (R1973/11-cl 2)
or LaSota /AF077761 (R1954/11-cl 1). This finding further supports the
notion of re-isolated vaccine strains and is clearly distinct from
described virulent genotype 2.II viruses, detected in ND outbreaks in
2006 in Egypt (Mohamed et al., 2011). All
viruses from the year 2006 (NDV/chicken/Egypt/2-4/2006; FJ969393,
FJ969394, FJ969395) belong to genotype 2.II but are of velogenic
pathotype with ICPI values between 1,6 to 1.8 and a have corresponding
proteolytic polybasic cleavage site (RRQKR*FIG). The re-isolation of
vaccine type virus in line with earlier description
(Abolnik et al., 2004;
Nagy et al., 2020) and it is conceivable,
that emergency vaccination in flocks with suspected ND might lead to
such double infections. Those circumstances have been considered by the
OIE (OIE, 2018) when defining criteria
for virulence: The definition includes the sequence information of the
proteolytic cleavage site, but has the amendment “Failure to
demonstrate the characteristic pattern of amino acid residues as
described above would require characterization of the isolated virus by
an ICPI test”. In the brain multicycle replication is restricted to
virulent NDV due to the cleavability of the F-protein
(Nagai et al., 1976;
Rott, 1979) and thus brain passage lead
to enrichment of virulent NDV. In case of contradicting results as
described by others (Nagy et al., 2020;
Tan et al., 2008) sequencing of
re-isolated virus from the brain is indicated.
The failure to demonstrate antigenic escape in genotype 2.XXI precursor
and its genotype 2.VII.1.1 successor virus indicate that other factors,
maybe founded in regulatory viral genes, contribute to the assertiveness
of a specific virus population. Co-infections of vaccine type and wild
type NDV point to a conjecturable practice of emergency vaccination and
unrestricted handling of diseased poultry, thereby fostering ND
endemicity in Egypt. Apart from moecular epidemiologic studies on NDV
transmission dynamics combatting ND endemicity in Egypt certainly
requires improved herd and trading management.