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.