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\begin{document}

\title{Genomic and antigenic properties of Newcastle Disease virus genotypes
2.XX1 and 2.VII from Egypt do not point to antigenic drift as a driving
force of spread}



\author[1]{Mahmoud Naguib}%
\author[2]{Dirk Höper}%
\author[3]{Magdy El-Kady}%
\author[4]{Manal Afify}%
\author[5]{Ahmed Erfan}%
\author[4]{Hassanein Abozeid }%
\author[5]{Wafaa Hassan}%
\author[5]{Abdel-Satar A. Arafa}%
\author[5]{Momtaz shahein}%
\author[2]{Martin Beer}%
\author[2]{Timm Harder}%
\author[2]{Christian Grund}%
\affil[1]{Uppsala Universitet}%
\affil[2]{Friedrich-Loeffler-Institute Federal Research Institute for Animal Health}%
\affil[3]{Beni Suef University Faculty of Veterinary Medicine}%
\affil[4]{Cairo University}%
\affil[5]{Animal Health Research Institute}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is
endemic in poultry in the Middle East causing continuing outbreaks in
poultry populations despite efforts to vaccinate. In the past, genotype
2.XXI (former 2.VI) was present in poultry in Egypt but has been
replaced by genotype 2.VII. We investigated whether virus evolution
contributed to superseding, and focused on the antigenic sites within
the Heamagglutinin-Neuramindase (HN) spike protein. Full length
sequences of a NDV genotype 2.VII isolate currently circulating in Egypt
was compared to a genotype 2.XXI isolate that was present as
co-infection with vaccine type viruses (2.II) in an historical isolate
of the year 2011. Amino acid differences in the HN glycoprotein for both
2.XXI and 2.VII viruses amounted to 11,7\% and 11,9 \% compared to
LaSota vaccine type. However, mutations within the globular head (aa
126-570), bearing relevant antigenic sites, were underrepresented (aa
divergence of 8,8\% and 8,1 \% compared to 22,4\% and 25,6\% within the
fragment encompassing cytoplasmic tail, transmembrane part and stalk
regions (aa 1-125) for genotypes 2.XXI and 2.VII, respectively.
Nevertheless, reaction patterns of HN-specific monoclonal antibodies
revealed differences between vaccine type viruses and genotype 2.XXI and
2.VII viruses for specific epitopes. Accordingly, compared to Egyptian
vaccine type isolates and the LaSota vaccine reference strain, single aa
substitutions in 6 of 10 described neutralizing epitopes were found
within the attachment protein. However, the same alterations in
neutralization sensitive epitopes were present in old genotype 2.XXI as
well as in newly emerged genotype 2.VII isolates. In addition, isolates
were indistinguishable by polyclonal chicken sera raised against
different genotypes including vaccine viruses. These findings suggest,
that factors other than antigenic differences within the HN-protein
account for facilitating spread of genotype 2.VII while displacing
genotype 2.XXI viruses in Egypt.%
\end{abstract}%



\sloppy


\textbf{Genomic and antigenic properties of Newcastle Disease virus
genotypes 2.XX1 and 2.VII from Egypt do not point to antigenic drift as
a driving force of spread}

(Running title: Absence of antigenic drift in Newcastle Disease virus)

Mahmoud M. Naguib\textsuperscript{1, 2}, Dirk H\selectlanguage{ngerman}öper\textsuperscript{3},
Magdy F. Elkady\textsuperscript{4}, Manal A. Afifi\textsuperscript{5},
Ahmed Erfan\textsuperscript{2}, Hassanein H. Abozeid\textsuperscript{5},
Wafaa M. Hasan\textsuperscript{2}, Abdel-Satar Arafa\textsuperscript{2},
Momtaz Shahein\textsuperscript{2}, Martin Beer\textsuperscript{3}, Timm
C. Harder\textsuperscript{3}, Christian
Grund\textsuperscript{3,\textbf{\#}}

\textsuperscript{1}Zoonosis Science Center, Department of Medical
Biochemistry and Microbiology, Uppsala University, 751 21 Uppsala,
Sweden

\textsuperscript{2} National Laboratory for Veterinary Quality Control
on Poultry Production, Animal Health Research Institute, Agriculture
research centre, Giza 12618, Egypt

\textsuperscript{3} Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Institute of Diagnostic Virology, 17493
Greifswald Insel-Riems, Germany

\textsuperscript{4} Department of poultry disease, Faculty of Veterinary
Medicine, Beni-Suef University, Beni-Suef 62511, Egypt

\textsuperscript{5} Department of Poultry disease, Faculty of Veterinary
Medicine, Cairo University, Giza 11221, Egypt

\textsuperscript{\textbf{\#}} \textbf{Corresponding author:}

Christian Grund

OIE, FAO and National Reference Laboratory for Newcastle disease,
Suedufer 10, 17493 Greifswald Riems, Germany

Christian.Grund@fli.de

Key words: Newcastle disease; Newcastle disease virus; genotypes; Egypt;
evolution; antigen sites;

\textbf{Abstract}

Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is
endemic in poultry in the Middle East causing continuing outbreaks in
poultry populations despite efforts to vaccinate. In the past, genotype
2.XXI (former 2.VI) was present in poultry in Egypt but has been
replaced by genotype 2.VII. We investigated whether virus evolution
contributed to superseding, and focused on the antigenic sites within
the Heamagglutinin-Neuramindase (HN) spike protein. Full length
sequences of a NDV genotype 2.VII isolate currently circulating in Egypt
was compared to a genotype 2.XXI isolate that was present as
co-infection with vaccine type viruses (2.II) in an historical isolate
of the year 2011. Amino acid differences in the HN glycoprotein for both
2.XXI and 2.VII viruses amounted to 11,7\% and 11,9 \% compared to
LaSota vaccine type. However, mutations within the globular head (aa
126-570), bearing relevant antigenic sites, were underrepresented (aa
divergence of 8,8\% and 8,1 \% compared to 22,4\% and 25,6\% within the
fragment encompassing cytoplasmic tail, transmembrane part and stalk
regions (aa 1-125) for genotypes 2.XXI and 2.VII, respectively.
Nevertheless, reaction patterns of HN-specific monoclonal antibodies
revealed differences between vaccine type viruses and genotype 2.XXI and
2.VII viruses for specific epitopes. Accordingly, compared to Egyptian
vaccine type isolates and the LaSota vaccine reference strain, single aa
substitutions in 6 of 10 described neutralizing epitopes were found
within the attachment protein. However, the same alterations in
neutralization sensitive epitopes were present in old genotype 2.XXI as
well as in newly emerged genotype 2.VII isolates. In addition, isolates
were indistinguishable by polyclonal chicken sera raised against
different genotypes including vaccine viruses.

These findings suggest, that factors other than antigenic differences
within the HN-protein account for facilitating spread of genotype 2.VII
while displacing genotype 2.XXI viruses in Egypt.

\textbf{Key words}

Newcastle disease virus, Egypt, pathogenicity, antigenic variation,
vaccine.

\textbf{Introduction}

Newcastle disease (ND) is a highly contagious disease with significant
clinical impact and serious losses in poultry worldwide. The causative
agent, Newcastle disease virus (NDV) or avian paramyxovirus type-1
(APMV-1) is a species within the genus Avian ortho\emph{avulavirus 1} ,
a member of the family \emph{Paramyxoviridae}(Rima et al., 2019). The
genome is composed of a negative-sense, single-stranded RNA of
approximately 15 kb coding for at least seven proteins, in the 3' to 5'
order nucleocapsid protein (NP), phosphoprotein (P), V protein (V),
matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN)
and large RNA-dependent polymerase protein (L) (Steward et al.,
1993).The HN protein recognizes and attaches to sialic acid receptors on
the surface of permissive cells and mediates the fusion activity of the
F protein at the cell membrane for release of the nucleocapsid complex
into the cytoplasm (Lamb \& Parks, 2007).

Based on its pathogenicity, NDV is categorized into four groups with
increasing virulence from apathogenic, lentogenic, mesogenic and
velogenic (Alexander, 2000). The molecular basis for NDV pathogenicity
is associated with the amino acid sequence motif of the protease
cleavage site of the fusion protein, and the abilities of specific
cellular proteases to cleave this protein. Based on phylogenetic
analysis, NDV can be distinguished in two distinct classes, 1 and 2,
with one (1.I) and twenty one genotypes, respectively (2.I-2.XXI)
(Dimitrov et al., 2019). Whereas the majority of class 1 viruses are
avirulent and have their natural reservoir in aquatic wild birds (Kim et
al., 2007), class 2 comprises virulent viruses that are responsible for
the outbreaks in poultry and evolved over time(P. J. Miller et al.,
2010). Phylogenetic evidence suggests that in the decades prior to the
1970s, genotypes 2.II, 2.III and 2.IV were predominant in North America,
Asia and Europe, respectively. Only genotype 2.II comprises of
lentogenic, mesogenic and velogenic pathotypes (Czegledi et al., 2002;
Czegledi et al., 2003). and vaccines derived from lentogenic strains of
this genotype (Seal et al., 1996). Genotype 2.VI viruses emerged in
epizootics in the Middle East and Asia in the 1960s, while in the early
1970s genotype 2.V (most likely of South American origin) caused
outbreaks in North America and in Europe (Herczeg et al., 2001; Wehmann
et al., 2003). Later, genotype 2.VI viruses gave rise to strains adapted
to pigeons, the so-called pigeon type paramyxovirus (PPMV-1) (D. J.
Alexander et al., 1985). The new NDV nomenclature proposal (Dimitrov et
al., 2019) placed subgroups 2.VIai and 2.VIaii, encompassing the early
strains circulating in poultry (Chong et al., 2013), into the new
genotypes 2.XX and 2.XXI whereas PPMV-1 is forming the ``new'' genotype
2.VI with several sub-genotypes. Most recently, several sub-lineages of
genotype 2.VII and 2.VIII emerged in the Far East and spread to further
geographic areas (Herczeg et al., 1999). Genotype 2.VII is currently
predominant among velogenic NDV and can be further subdivided i.e. with
sub-lineages 2.VII.1.1 encompassing viruses that emerged in the 1990s in
the Far East, Europe and Asia, and the Middle East; while 2.VII.1.2
viruses evolved in China, and circulated in South and East Africa, as
well as in Israel and Pakistan (Dimitrov et al., 2019).

In Egypt, ND is endemic and despite extensive routine vaccination
programs implemented in the commercial poultry farm sectors, new cases
occur continuously, posing a threat to the national poultry industry. In
Egypt, ND was identified for the first time in 1948 (Daubney \& Mansy,
1948), and in the last few years NDV 2.II and 2.VII genotypes were
reported from the country (Radwan et al., 2013). Also, co-infections
with infectious bronchitis and avian influenza viruses were recorded in
Egypt (Moharam et al., 2019; Naguib et al., 2017; Samy \& Naguib, 2018).
This has raised concerns regarding the efficacy of ND vaccination
programs applied in the country. It has been hypothesized that, due to
accumulated mutations as seen previously with highly pathogenic avian
influenza virus (Grund et al., 2011), new NDV antigenic variants have
arisen, that have an advantage to spread within a vaccinated population.
In consequence, viruses that supersede previous virus populations should
have a gain in fitness, here, mutations within antigenic relevant sites.
To test this hypothesis, four isolates representing genotype 2-II (n=2),
the old genotype 2.XXI (former 2.VIa; n=1) and the currently circulating
genotype 2.VII.1.1 in Egypt (n=1) were genetically and antigenically
analyzed, with special focus on the viral glycoproteins, that are
facilitating receptor mediated virus uptake and are the main target for
protective immune responses.

\textbf{Material and Method}

\emph{Sample collection and virus identification}

Samples (oropharyngeal and cloacal swabs, trachea or kidneys) were
obtained from outbreaks of systemic disease in ten different commercial
chicken farms in Egypt during 2011-2014 as shown in table 1. Samples
were collected by the Reference Laboratory for Quality Control on
Poultry production (RLQP)-Animal Health Research Institute (AHRI) and
Beni-Suef University, Egypt, and shipped to the
Friedrich--Loeffler-Institute (FLI), Germany. Identification of NDV was
based on real-time reverse transcriptase polymerase chain reaction
(RT-qPCR) using the Biorad CFX1000 Real-Time PCR system. Briefly, RNA
was extracted from collected samples by QiaAmp viral RNA extraction kit
(Qiagen, Hilden, Germany) as recommended by the manufacturer's
instructions. NDV nucleic acid was detected by SuperScript® III One-Step
RT-PCR using primers and probe specific to the M gene (Wise et al.,
2004). Positive samples were subjected to another RT-qPCR to
differentiate lentogenic from velogenic viruses based on the sequence of
fusion protein cleavage site (FPCS) using primers and probe specific for
the M and F genes (Aldous et al., 2001; Moharam et al., 2019).

\emph{Virus isolation}

Hepatocellular carcinoma epithelial cells (LMH) (Kawaguchi et al., 1987)
were cultured with 2 \selectlanguage{greek}µ\selectlanguage{english}g TCPK Trypsin/mL in T25 culture flasks and
infected with 0.5 mL of the NDV-positive samples. Cells were incubated
at 37 \selectlanguage{ngerman}°C until giant cell formation was observed or for up to 7 days
before initiating a second passage following a freeze/thaw cycle.
Further confirmation using hemagglutination (HA) and hemagglutination
inhibition (HI) tests were conducted on the supernatant culture medium
(OIE, 2012). Isolated viruses were aliquoted and kept at -80
\textsuperscript{0}C until further use.

\emph{Plaque purification}

Plaque assay was used to obtain purified virus clones for selected
samples showing unequivocal results with pathotype-specific RT-qPCRs.
Briefly, different virus dilutions were incubated on confluent LMH cell
monolayers cultured in six-well plates overnight at 37°C. Then, virus
inoculum was removed, cell layers washed carefully twice with sterile
PBS and overlaid with modified Eagle's medium containing 1.8 \% agar.
Infected cells were incubated at 37 °C and 55\% humidity for three days.
Finally, selected plaques were picked and propagated on LMH cells as
described above for further characterization.

\emph{Genetic characterization and phylogenetic analysis}

Amplification of a 698 bps fragment spanning part of the NP and the F2
gene of NDV was performed using SuperScript III One-Step RT-PCR system
(Invitrogen, Carlsbad, CA, USA) following published protocols (Aldous et
al., 2003). Amplicons were size-separated by agarose electrophoresis,
excised and purified from gel using the QIAquick Gel Extraction kit
(Qiagen, Hilden, Germany). Purified PCR products were used directly for
cycle sequencing reactions using BigDye Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction
products were purified using NucleoSeq kit (Macherey-Nagel, Düren,
Germany) and sequenced on an ABI PRISM® 3100 Genetic Analyzer (Life
Technologies, California, USA).

Assembly of the obtained sequences was performed using the Geneious
software, version 9.0.5 (Kearse et al., 2012). Alignment and identity
matrix analyses were conducted using MAFFT (Katoh \& Standley, 2013) and
BioEdit (Hall, 1999). Sequences generated in this study were submitted
to the GenBank NCBI (National Center for Biotechnology Information)
database (http://www.ncbi.nlm.nih.gov/) and assigned accession numbers
shown in Table 1. Reference sequences of NDV genotypes (Diel et al.,
2012; Snoeck et al., 2013) required for further analyses were retrieved
from the GenBank database. Phylogenetic analyses were performed and
trees were constructed using the IQ-tree software version 1.1.3 (Minh et
al., 2013; Nguyen et al., 2014) based on maximum likelihood analysis of
phylogenetic relationship after selection of the best fit substitution
model. Finally, trees were viewed and edited using FigTree v1.4.2
software (http://tree.bio.ed.ac.uk/software/figtree/\emph{)} and
Inkscape 0.51.

\emph{Full Genome Sequencing}

Full genome sequences of four successfully isolated NDV strains, namely,
R1954-11-cl-1, R1973-11-cl-2, R1973-11-cl-4 and AR2178-14, representing
three genotypes previously reported in Egypt, were carried out. Briefly,
RNA was purified from isolates using TRIzol LS reagent
(LifeTechnologies, Darmstadt, Germany) and an RNeasy mini kit (Qiagen,
Hilden, Germany) with on-column DNase digestion according to the
manufacturer's instructions. Conversion of RNA into double-stranded DNA
was performed using a cDNA synthesis system (Roche, Mannheim, Germany).
Library construction was done as previously described (Juozapaitis et
al., 2014). Sequencing was performed on an Illumina MiSeq Instrument
using the MiSeq reagent kit version 3 (Illumina, San Diego, CA, USA).

Assembly of the sequence data was done using the Genome Sequencer
version 2.6 software suite (Roche), and NDV-related contigs were
identified with BLASTn (BLASTn;
http://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained full-length NDV
sequences have been submitted to GenBank (Table 1). Further, the open
reading frame (ORF) analysis and the annotation of the genome were
carried out using the Geneious software.

Phylogenetic analyses were carried out for both of F-gene (partial/full)
and the whole genome sequence separately using references viruses as
mentioned in the previous section.

\emph{Pathotyping}

Intracerebral pathogenicity index (ICPI) was determined following
European guidelines (CEC, 1992). Ten one-day-old SPF chickens were
inoculated intracerebrally with 0.05 ml of 10\textsuperscript{-1}
diluted virus stock (HA \textgreater{} 32) and examined daily for 8
days. The ICPI is calculated by scoring each bird every day from 0
(normal), 1 (sick) and to 2 (dead). Based on this score NDV-isolates are
considered lentogenic (-0.7), mesogenic (\textgreater{}0,7-1.3) or
velogenic (\textgreater{}1,5) (Alexander, 1998). Animal experiments were
carried out in accordance with the legally approved protocol (MV-LALLF-
/7221.3-1.1-030/13). Specific pathogen free White leghorn chickens
purchased from Lohmann Animal Health, Cuxhaven, Germany, were raised in
isolation units at FLI until age of inoculation. Infection and
immunization experiments were performed in in separated cages at animal
biosafety level-3 (BSL-3). Feed and water were provided daily.

\emph{Preparation of polyclonal anti-NDV chicken antisera}

Four reference NDV strains, clone30, Ulster, R151/94, and R1468/12,
representing the avian orthoavulavirus-1, 2.II, 2.VI (PPMV-1) and
2.VII.1.1, respectively, were selected for preparation of polyclonal
anti-NDV chicken antiserum in the FLI animal facility. Viruses were
first inactivated by betapropiolactone (Sigma; 0,05\%,) treatment and
0.5 mL of each virus was injected in four 4-week-old-SPF White Leghorn
chickens via subcutaneous route after mixing with Freund's complete
adjuvant (1:1). A second immunization shot was given after 4 weeks.
Collection of serum was performed at 7 days after the booster
immunization, followed by inactivation at 56°C for 30 min and stored at
-20 °C until further use. Immunizations were carried out in accordance
with the legally approved protocol (MV-LALLF- 7221.3-2.5-010/10).

\emph{Antigenic characterization}

Antigenic analysis of three Egyptian NDVs AR2178-14, R1973-11-cl4 and
R1954-11-cl6 as well as of another five reference strains was performed
by hemagglutination inhibition (HI) assay, as described (CEC, 1992). In
addition to a panel of four polyclonal sera (see previous section) and
five HN-specific monoclonal antibodies were used (mAb 617/161, mAb U85,
mAb 7D4 were a generous gift from AVLA-Weybridge and mAb 39-2, mAb 10
were a gift from Bernd Köllner, FLI)

\textbf{Results}

\emph{RT-qPCR, virus isolation and purification}

Molecular pathotyping based on RT-qPCR using the primers and probes
specific for avirulent and virulent pathotypes (RT-F-qPCR) revealed that
two Egyptian NDV isolates of the year 2011 were positive for lentogenic
as well as virulent pathotypes, indicating mixed infection. The
consensus sequence of the proteolytic cleavage site within the
F\textsubscript{2}-protein of one mixed isolate (R1954-11) represents a
polybasic recognition motive, characteristic for virulent
meso-/velogenic pathotypes (. This would be in agreement with the
obtained ICPI of 0.91, representing a mesogenic pathotype. In contrast,
the sequence of the second mixed isolate R1973/11 represents a cleavage
site of lentogenic vaccine type viruses but had an ICPI of 1.88, clearly
identifying the isolate as velogenic. By plaque cloning, clones with
mono- (R1954/-11- cl1; R1973/11-cl 2) or polybasic cleavage sites
(R1954/11-cl 6; R1973/11-cl 4) could be obtained from both samples. For
these purified clones, unambiguous reactivity patterns in
pathotype-specific RT-qPCRs were consistent with their sequences of the
proteolytic cleavage site and their ICPI values even though the ICPI
value of R1973-11-cl 2 was slightly above the threshold of 0.7 between
lentogenic to mesogenic strains.

\emph{Genetic and phylogenetic characterization}

Sequences of the F2-fragment, including the proteolytic cleavage site
were used for initial phylogenic characterization (358 nt). BLAST tools
indicated highest homology for isolate AR2178/2014, with Egyptian Avian
avulavirus-1/168-2012 (Genbank accession: MN381174) with nt and aa
identities of 99\% and 99\%, respectively, clustering in phylogenetic
analysis with genotype 2.VII.1.1 viruses (supplementary Fig 1.). In
contrast, virulent NDV clones from 2011 (R1954/2011-cl 6; R1973/2011-cl
4) showed highest identities to EG-14/90 (Genbank accession: DQ096604)
and chicken/Nigeria/2006 (Genbank accession: MH092825) respectively The
lentogenic clone R1954/2011-cl 1 matched 100 \% with vaccine strain
LaSota (Genbank accession: AF077761), while R1973/2011-cl 2 matched with
vaccine strain Hitchner B1 (Genbank accession: JN872151). When sequence
collection of the genotyping group (Dimitrov et al. 2019) were used to
create a first phylogenetic analysis (S1), viruses grouped to genotype
2.VII.1.1 (AR2178/2014), 2.XXI (R1954/2011-cl 6; R1973/2011-cl 4) and
2.II (R1954/2011-cl 1; R1973/2011-cl 2). While genotype 2.VII.1.1
viruses are circulating in Egypt from 2012 up to 2019 (Fig. S1), no
further sequences for NDV genotype 2.XXI have been reported for chickens
after 2011. However subsequent viruses from genotype 2.XXI belong to
sub-genotype 2. XXI.1.1 and are derived from pigeons.

\emph{Full genome sequence analysis}

To verify and further analyze selected NDV isolates, full genome
sequences were generated for both vaccine type clones (R1954/2011-cl 1,
R1973/2011-cl 2) as well as for genotype 2.XXI (R1973/2011-cl 4) and
2.VII.1.1 (AR2178/2014) isolates. The genomes obtained for the
lentogenic NDV clones R1954/2011-cl 1 and R1973/2011-cl 2 were 15.135
and 15.150 nt long, whereas the genomes of the virulent strains
AR2178/2014 and clone R1973/2011-cl 4 were 15,179 and 15,165 nt long
with a 6-nt-insertion ACCCCC or CCTCTC respectively in the untranslated
region (UTR) downstream of the NP gene.

The two NDV strains of R1954/2011-cl 1 and R1973/2011-cl 2 showed 99\%
nt similarity with the NDV strain LaSota C5 (accession: KC844235) (Table
2). On the other hand, the velogenic NDV strain AR2178/2014 showed the
highest nt sequence similarity of 99\% to the Chinese NDV strain
SD04/2011 (accession: JQ015296), whereas strain R1973/2011-cl 4 showed
94\% nt similarity with the Japanese NDV strain Osaka/2440/1969
(accession: AB853926).

Phylogenic analysis of the full F gene (Fig. 1) as well as the whole
genome sequence (Fig. 2) confirmed the phylogrouping obtained by partial
F-gene analysis (Fig. S1). Genome organization of the four fully
sequenced NDV strains was found to be identical to other Avian
orthoavulavirus -1, containing six genes in the order of
3\selectlanguage{english}'-NP-P-M-F-HN-L-5\selectlanguage{english}', encoding at least seven proteins; N, P, V, M, F, HN
and L. Analysis of the F protein of the four NDV strains revealed that
three strains, namely, R1954/2011-cl 1, R1973/2011-cl 2 and AR2178/2014
(genotype 2.II, and 2.VII.11, respectively) possessed six potential
N-glycosylation sites (NGS); five within the ectodomain at amino acid
(aa) positions 85, 191, 366, 447, and 471 and one in the cytoplasmic
domain at position 542. Strain R1973/2011-cl 4 (genotype 2.XXI)
possessed the same five ectodomain NGS and an additional one at position
aa 4 (https://prosite.expasy.org/PDOC00001 ).

The HN proteins of the two NDV strains R1973/2011-cl 4 and AR2178/2014
(genotype 2.XXI and 2.VII.1.1, respectively) were 571 aa long, which is
6 aa shorter than those of the NDV strains of genotype 2.II, namely,
R1954/2011-cl 1 and R1973/2011-cl 4 (577 aa). The HN protein of all
genotypes possessed five potential NGS. Four NGS at positions 119, 341,
433 and 481 were common in the four NDV strains, whereas NDV strains
R1973/2011-cl 4 and AR2178/2014 (genotype 2.XXI and 2.VIIb,
respectively) possessed an additional NGS at site 508, and strains
R1954/2011-cl 1 and R1973-11-cl 2 (genotype 2.II) at position 538.

Comparing the level of homology of the different isolates to the La Sota
vaccine strain (Accession No. AF077761) confirms the close relation in
between the genotype 2.II viruses. Regarding genotype 2.XXI and 2.VII,
both genotypes accumulated mutations compared to La Sota vaccine strain,
but these changes were present for both viruses to a comparable level
with homology of 81.0 to 85.5\%. For the different genes, no striking
differences are evident. Comparing the amino acid (aa) composition of
genotype 2.XXI and 2.VII.1.1, the homology to 2.II strain La Sota is
higher than on the level of nucleotides, with 92 to 87.9 \% for NP, M,
F, HN and L-protein. It is striking that the homology of the P-protein
was considerably lower than the other genes with 80.1 and 81.9\% for
genotype 2.XXI and 2.VII.1.1 respectively. When focusing on the HN
protein, responsible for receptor mediated binding, our data reveal
overall aa differences of 11,7\% and 11,9 \% for 2.XXI and 2.VII.1.1
viruses respectively compared to LaSota vaccine type (Table.3).
Mutations accumulated within the protein fragment encompassing the tail,
transmembrane part and stalk region (aa 1-125; 22,4\% and 25,6\%),
whereas the globular head (aa 126-570), bearing relevant antigenic
sites, was underrepresented and had an aa divergence of only 8,8\% and
8,1 \% for genotypes 2.XXI and 2.VII.1.1 respectively. Among the aa in
the globular head, 21 of 39 and 35 substitutions were identical for both
2.XXI and 2.VII.1.1 viruses. These changes include 6 single point
mutations in sites that were described to be part of neutralization
sensitive epitopes (Y203H, N263K, N347K, E494D, G495E and V514E) of the
HN-protein (Table 3), i.e. site site 23; site 3, site I, site12, site
II, site 2 (Iorio et al., 1989; Iorio et al., 1991) (Iorio). In an
additional six positions both viruses had changes compared to LaSota
(aa, 266, 288, 310, 433, 443, 509) but differed in aa composition.
Unique changes were evident on 9 and 10 positions for genotype 2.XXI and
2.VII.1.1 viruses respectively. None of those isolate-specific sites
affected known functional sites directly. For the vaccine type isolates
only R1973/2011-cl 1 had 5 alterations, that were all located within the
globular head of the HN-protein, with mutations in epitope 23 (Y203H)
and NA binding site (A502V) that were identical for the genotype 2.XXI
and 2.VII.1.1 viruses.

\emph{Antigenic characterization}

By cross hemagglutination inhibition (HI) assays (Table 4) reaction
patterns of sera directed against clone 30 (2.II) or Ulster (2.I.2) and
the Egyptian isolates of genotype 2.II were indistinguishable.
Reactivity even with a phylogenetically distant class 1 APMV-1 isolate
(R2919/06) was in the range of 2 log\textsubscript{2} steps. In
contrast, reactivity of a serum raised against another genotype
2.VII.1.1 virus (R1468/12) was considerably lower with genotype 2.II and
2.I viruses (3 log2) as well as with the class 1 virus (4 log2).
However, genotype 2.VII.1.1 specific serum did not distinguish between
Egyptian genotype 2.VI and 2.VII.1.1 viruses. In addition, a serum
raised against classical PPMV-1 virus (R151/94) assigned to genotype
2.VI did show equal reactivity to both genotype 2.XXI chicken isolates,
but reactivity was decreased to genotype 2.II and 2.I and class 1 virus
and to a lesser extent to genotype 2.VII.1.1 viruses. Reactivity
profiles obtained by HI with a panel of mAbs, further highlighted
antigenic differences between PPMV-1 and other genotype 2.XXI strains
(R1973/2011-cl 4 and R1954/2011-cl 6). A second set of mAbs, reactive
with vaccine type genotype 2.II viruses varied in their reactivity
profile: whereas mAb U85 recognized genotype 2.II, 2.I and R1468/12
(2.VII.1.1) viruses, mAb 7D4 was specific only for vaccine type 2.II
viruses, and mAb 10 recognized an epitope present in genotype 2.II and
2.I viruses. These data strongly suggested, that epitope specific
difference existed, but that they were of minor importance for
recognition by polyclonal sera that comprise antibodies directed against
multiple epitopes.

\textbf{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 (Ujvari 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; Ujvari 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
(~\textsuperscript{112}R/K-R-Q-K/R-R*F\textsuperscript{117}) and
avirulent NDV strains
harbor~\textsuperscript{112}G/E-K/R-Q-G/E-R*L\textsuperscript{116}~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; Romer-Oberdorfer 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.

\textbf{Declarations}

\emph{Competing interests}

The authors declare that they have no competing interests.

\emph{Acknowledgment}

The authors thank Cornelia Illing, FLI, Germany, for excellent technical
support. We are grateful to colleagues and co-workers at NLQP and
Beni-Suef university, Egypt, for helping in field samples collection.

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\textbf{Table 1: Egyptian NDV isolates included in the study}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/378915/articles/495316-genomic-and-antigenic-properties-of-newcastle-disease-virus-genotypes-2-xx1-and-2-vii-from-egypt-do-not-point-to-antigenic-drift-as-a-driving-force-of-spread}

\textbf{Table 2:} Similarity comparisons of nucleotide and amino acid
sequences of ND viruses sequenced in this study and the LaSota vaccine
strain (AF077761).

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/378915/articles/495316-genomic-and-antigenic-properties-of-newcastle-disease-virus-genotypes-2-xx1-and-2-vii-from-egypt-do-not-point-to-antigenic-drift-as-a-driving-force-of-spread}



\textbf{Table 3: Amino acid mutations of Egyptian viruses within the
HN-protein}

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/378915/articles/495316-genomic-and-antigenic-properties-of-newcastle-disease-virus-genotypes-2-xx1-and-2-vii-from-egypt-do-not-point-to-antigenic-drift-as-a-driving-force-of-spread}

\textbf{Table 4.} Antigenic characterization of the NDV strains isolated
in this study and NDV reference viruses as well as anti-F mAbs\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
& & & & \textbf{Virus - Antigen} & & & &\tabularnewline
\midrule
\endhead
& 1 & 2.I & 2.II & 2.VI & 2.XXI & 2.XXI & 2.VII & 2.VII\tabularnewline
\textbf{Serum} & \textbf{R2919/06} & \textbf{Ulster} & \textbf{Clone 30}
& \textbf{R 151/1994} & \textbf{R1954/2011-cl 6} & \textbf{R1973/2011-cl
4} & \textbf{R1468/2012} & \textbf{AR 2178/2014}\tabularnewline
\textbf{a-Ulster (2.I)} & \textbf{10} & \textbf{10} & \textbf{11} &
\textbf{11} & 11 & 11 & \textbf{10} & \textbf{10}\tabularnewline
\textbf{a-clone30 (2.II)} & \textbf{6} & \textbf{7} & \textbf{8} &
\textbf{7} & 8 & 9 & \textbf{8} & \textbf{8}\tabularnewline
\textbf{a-R151/94 (PPMV-1)} & \textbf{5} & \textbf{6} & \textbf{6} &
\textbf{8} & 8 & 9 & \textbf{6} & \textbf{7}\tabularnewline
\textbf{a-R1468/12 (2.VII)} & \textbf{6} & \textbf{7} & \textbf{7} &
\textbf{8} & \textbf{10} & \textbf{11} & \textbf{10} &
\textbf{9}\tabularnewline
\textbf{mAb 617/161} & \textbf{3} & \textbf{\textless{}1} &
\textbf{\textless{}1} & \textbf{11} & \textbf{1} & \textbf{2} &
\textbf{\textless{}1} & \textbf{\textless{}1}\tabularnewline
\textbf{mAb 39} & \textbf{3} & \textbf{\textless{}1} &
\textbf{\textless{}1} & \textbf{6} & \textbf{\textless{}1} &
\textbf{\textless{}1} & \textbf{\textless{}1} &
\textbf{\textless{}1}\tabularnewline
\textbf{mAb U85} & \textbf{4} & \textbf{8} & \textbf{11} & \textbf{1} &
\textbf{2} & \textbf{4} & \textbf{7} & \textbf{1}\tabularnewline
\textbf{mAb 7D4} & \textbf{3} & \textbf{1} & \textbf{11} & \textbf{1} &
\textbf{1} & \textbf{2} & \textbf{2} & \textbf{1}\tabularnewline
\textbf{mAb 10} & \textbf{1} & \textbf{5} & \textbf{8} &
\textbf{\textless{}1} & \textbf{\textless{}1} & \textbf{\textless{}1} &
\textbf{\textless{}1} & \textbf{\textless{}1}\tabularnewline
[?]HI to homologe virus & & & & \textbf{[?] 2} & \textbf{[?]2} & \textbf{[?]4} &
\textbf{[?]6} &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1.} Phylogenetic tree of ND viruses based on nucleotide
sequence of full F gene.

Phylogenetic tree was based on maximum likelihood calculations (IQTree
software) under the best fit model (model: GTR+F+I+G4) according to the
Bayesian Information Criterion (BIC). Numbers at nodes represent
measures of robustness based on an ultrafast bootstrap approach
implemented in IQTree. The sequences of other reference strains as well
as all publicly available Egyptian NDV were obtained from GenBank.
Divisions and genotypes are designated according to Dimitrov et al.,
2019. Egyptian viruses are colored in red, and black circle is infront
of viruses of this study.

\textbf{Figure 2.} Phylogenetic tree of NDV viruses based on whole
genome sequences.

Phylogenetic tree was based on maximum likelihood calculations (IQTree
software) under the best fit model (model: GTR+F+I+G4) according to the
Bayesian Information Criterion (BIC). Numbers at nodes represent
measures of robustness based on an ultrafast bootstrap approach
implemented in IQTree. The sequences of other reference strains as well
as all publicly available Egyptian NDV were obtained from GenBank.
Divisions and genotypes are designated according to Dimitrov et al.,
2019. Egyptian viruses are colored in red, and black circle is in front
of viruses of this study.

\textbf{Figure S1.} Phylogenetic tree of ND viruses based on partial
sequence of the F gene (375 bp) spanning the cleavage site.

Phylogenetic tree was based on maximum likelihood calculations (IQTree
software) under the best fit model (model: TIM2e+G4) according to the
Bayesian Information Criterion (BIC). Numbers at nodes represent
measures of robustness based on an ultrafast bootstrap approach
implemented in IQTree. The sequences of other reference strains as well
as all publicly available Egyptian NDV were obtained from GenBank.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1-radial-Full-F-101028/Figure-1-radial-Full-F-101028}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2-whole-genome-201028/Figure-2-whole-genome-201028}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
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