Received: 18 March 2022 Revised: 14 June 2022 Accepted: 4 July 2022

DOI: 10.1111/tbed.14654

S H O R T C O M M U N I C AT I O N

Candidate ‘Avian orthoreovirus B’: An emerging waterfowl

pathogen in Europe and Asia?

Renáta Varga-Kugler1 Szilvia Marton1 Ákos Thuma2

Katalin Szentpáli-Gavallér2 Ádám Bálint2 Krisztián Bányai1,3

1
Pathogen Discovery Group, Veterinary
Medical Research Institute, Budapest,
Hungary
2
Veterinary Diagnostic Directorate, National
Food Chain Safety Office, Budapest, Hungary
3
Department of Pharmacology and Toxicology,
University of Veterinary Medicine, Budapest,
Hungary
Correspondence
Krisztián Bányai, Veterinary Medical Research
Institute, Hungária krt. 21., H-1143 Budapest,
Hungary.
Email: bkrota@hotmail.com
Abstract
A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019,
Hungary. The affected flock experienced a marked decrease in egg production.
Histopathological lesions were seen in the oviduct and in the lungs of birds sent
for diagnostic investigation. The fusogenic agent was characterized as an orthore-
ovirus by viral metagenomics. The assembled viral genome was composed of 10
genomic segments and was 23,433 nucleotides (nt) in length. The study strain, desig-
nated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity
with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%–
72.33%) as well as with representative strains of neoavian orthoreoviruses (nt,
40.07%–68.23%). On the contrary, the study strain shared 86.48%–95.01% pairwise
nt sequence identities with recent German and Chinese reovirus isolates, D2533/6
and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl
pathogens on a monophyletic branch, indicating a common evolutionary origin of
Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the
past few years. The finding that a candidate new orthoreovirus species, tentatively
called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in mori-
bund ducks seems an alarming sign that needs to be better evaluated by extending
laboratory diagnosis of viral pathogens in countries where the waterfowl industry is
important.

KEYWORDS
Hungary, Pekin duck, phylogenetic analysis, reovirus

1 INTRODUCTION
Reoviruses of birds are frequently detected and isolated from domestic
poultry with various morbidities. Moreover, some reovirus-associated
diseases are a major cause of economic losses for the poultry industry.
Since its first description in 1950 in Africa (Kaschula, 1950), reovirus
infection of waterfowl has been detected in many parts of the world,
mainly Europe and Asia.
Based on biological and genetic properties, waterfowl-origin
reoviruses (WRVs) are classified into two categories, the classical and
novel WRVs. Thus far, classical WRVs have been identified only in Mus-
covy ducks (Cairina moschata) and geese (Anser anser domestica) and
typically affect young birds exhibiting lethargy, weakness, diarrhoea,
later lameness and stunting; the mortality rate remains relatively low
(under 20%) (Heffels-Redmann et al., 1992; Malkinson et al., 1981;
Palya et al., 2003). In late 1990s, novel WRVs emerged in China; this
novel variant is associated with more severe symptoms, a higher (up
to 50%) mortality rate and a broader host range, such as Muscovy
duck, Pekin duck (Anas platyrhynchos domestica), mallard duck (Anas
platyrhynchos) and goose (Chen et al., 2012; Huang et al., 2022; Liu

e3386 © 2022 Wiley-VCH GmbH. wileyonlinelibrary.com/journal/tbed Transbound Emerg Dis. 2022;69:e3386–e3392.


VARGA-KUGLER ET AL .
et al., 2011; Luo et al., 2021; Yu et al., 2014; Yun et al., 2012). In addition
to the classical and the novel WRVs, some observations suggest that
waterfowl species are susceptible to infection with reoviruses of
galliform birds (Kim et al., 2022). Finally, researchers from Europe
and China independently identified novel waterfowl-associated
reoviruses. These unclassified RVs are currently represented only by
two strains, both isolated from young Pekin ducks (Cao et al., 2019;
Farkas et al., 2018).
WRVs possess non-enveloped virion particles that enclose the 10-
segmented dsRNA genome (large: L1–L3; medium: M1–M3; small:
S1–S4) (Matthijnssens et al., 2022). All currently known and classified
WRVs are members of the Avian orthoreovirus species (Orthoreovirus
genus). Genetic differences have been reported between classical and
novel WRVs. Most importantly, in classical WRVs, the S4 segment is
bicistronic and encodes the p10 and σC (cell attachment outer fibre)
proteins but lacks the FAST protein coding gene, which is responsible
for giant cell formation of fusogenic orthoreoviruses in cell cultures. In
novel WRVs, the S1 segment, the counterpart of S4 of classical WRVs, is
tricistronic; it encodes the p17 and the σC proteins, as well as the FAST
protein (Cao et al., 2019; Farkas et al., 2014, 2018; Huang et al., 2022).
In this study, we report the isolation and characterization of a
waterfowl-associated fusogenic reovirus strain, which together with
novel German and Chinese duck reoviruses may represent a new
orthoreovirus species pathogenic to domestic waterfowl.
2 MATERIALS AND METHODS
2.1 Background
In 2019, a Pekin duck breeder flock in the eastern part of Hungary
reported reduced egg production among 31-week-old ducks with a
rapid spread of clinical manifestations between duck sheds. The rate
of egg production decline was estimated at 70%–90% in all affected
houses.
2.2 Histology
Tissue samples collected for histological examination were fixed in 10%
buffered formalin. Four-micrometre thick sections of formalin-fixed
and paraffin-embedded tissue samples were stained with haematoxylin
and eosin and examined by light microscopy.
2.3 Virus detection, isolation
Consistent with the histopathological alterations, influenza virus-,
adenovirus-, flavivirus- and pneumovirus-specific molecular detection
assays were performed (Bäyon-Auboyer et al., 1999; Bäyon-Auboyer
et al., 2000; Eiden et al., 2010; Shin et al., 2000). In addition, samples
taken from birds were inoculated on duck origin AGE1.CR.pIX cells and
the cytopathic effect was monitored on a daily basis.
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e3387
2.4 Viral metagenomics
After one freezing/thawing cycle, 250 μl of cell culture supernatant
was used for the extraction of viral RNA using TRI Reagent (Molecular
Research Center) according to the manufacturer’s recommendations.
Extracted RNA was processed as described in detail previously (Bali
et al., 2021). For unbiased sequence-independent amplification of the
purified viral nucleic acid, random hexamer primed reverse transcrip-
tion coupled with PCR was carried out. Then, the cDNA library was
prepared using the Illumina® Nextera XT DNA Library Preparation Kit
(Illumina) and sequencing was performed on an Illumina® NextSeq 500
platform using a mid-output 150-cycle flowcell.
2.5 Computer analysis
Sequence reads generated by Illumina sequencing were assembled
by CLC Genomics Workbench version 7 (http://www.clcbio.com). A
combination of de novo assembly and reference sequence mapping
was utilized where we used the GenBank entry of strain D2533/6
(acc.no. MH520085–MH520094) based on initial evaluation of
the obtained contigs. A total of 49,163,102 reads were generated,
out of which 32,236,031 million reads mapped onto the reference
genome at ∼220,000× sequencing depth. Homologous genes among
sequences deposited in GenBank were identified by BLASTn and
BLASTx algorithms (Altschul et al., 1990). Codon-based multiple
sequence alignments were generated using the Muscle algorithm
within the Geneious Prime software (Kearse et al., 2012). Phylogenetic
analysis was performed and sequence identity values were calculated
for each genome segment using the MEGA6 package (Tamura et al.,
2013). Phylogenetic trees were constructed using the maximum
likelihood method, and their confidence was tested by bootstrap
analysis.
2.6 Data availability
The complete genome sequence of strain Reo/HUN/DuckDV/2019
was deposited into GenBank under the accession numbers
OM908476–OM908485.
3 RESULTS
3.1 Case description and virus detection
Decreased egg production was observed in a 31-week-old breeder
flock of Pekin ducks in the eastern region of Hungary. Gross pathology
showed systemic amyloidosis, histopathological examination revealed
lymphocytic interstitial pneumonia and lymphocytic inflammation of
the oviduct in affected birds (Figure 1). Influenza virus, adenovirus,
flavivirus and pneumovirus infections were excluded by molecular
detection methods. Duck embryo liver cell culture showed syncytium

e3388
F I G U R E 1 (a) Mild lymphohistiocytic infiltration in the muscle
tissue of the oviduct around the blood vessels. H-E staining 200×. (b)
Lymphohistiocytic interstitial pneumonia. H-E staining 200×. (c)
Syncytium in cell culture monolayer 72 h post infection
formation upon inoculation of homogenized organ samples (Figure 1).
Next, we carried out viral metagenomics using the nucleic acid extract
of cell culture supernatant. With this approach, large contigs obtained
by the assembly of >30 million homologous sequence reads identified
the isolated virus as an orthoreovirus homologous to but differ-
ent from representatives of species Avian orthoreovirus and Neoavian
orthoreovirus.
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VARGA-KUGLER ET AL .
3.2 Characterization of virus isolate
Genomic organization of the study strain was similar to that of other
ARVs. The shared features included the range of genome size (23,433
nt), the 5’ and 3’ termini of the genome segments, as well as the gene
structure and protein coding capacity (Table 1).
The study strain shared low-to-moderate sequence identity values
with representatives of the ARVs isolated from domestic poultry
(Table 2). When compared to different chicken and turkey origin
ARVs and WRVs, nucleotide (nt) sequence identity values ranged
from 38.90% to 72.33%. Similarly, in comparison with representative
members of the newly accepted avian origin orthoreovirus species,
Neoavian orthoreovirus (NeARV), the nt sequence identity values fell
between 40.07% and 68.23%. These values are below the uniform
gene-wise nt identity cut-off value (i.e., 75%) currently used as a
demarcation criterion for identical species within the Orthoreovirus
genus (Matthijnssens et al., 2022). In the case of protein sequence
similarities, Reo/HUN/DuckDV/2019 shared low-to-moderate aa
identity with ARVs (including WRVs) and NeARV (range, 41.13%–
84.79%). Each value fell below the cut-off values, except for the σB
protein sequence (ARV, 54.88%–61.62%, NeARV, 37.37%–38.05%),
where the study strain shared somewhat greater similarity to ARVs
(including WRVs) than expected based on other pairwise similarity
values.
On the other hand, comparing the study strain with strains D2533/6
and Ych isolated from Pekin ducklings in Germany and China, respec-
tively, high-sequence identity values were observed (nt, 86.48%–
95.01%; aa, 94.21%–99.21%), indicating that these three strains
belong to the same orthoreovirus species (Cao et al., 2019; Farkas et al.,
2018).
Phylogenetic trees based on the coding sequence of individual
segments showed that Reo/HUN/DuckDV/2019 constituted a mono-
phyletic clade with D2533/6 and Ych and these three strains were only
distantly related to other known orthoreoviruses of poultry (including
chicken, turkey and waterfowl origin strains) as well as the representa-
tive members of NeARVs (Figure 2, Supplementary File). Furthermore,
the three novel waterfowl-associated reovirus strains formed a com-
mon phylogenetic clade that was basal to the major clade of species
Avian orthoreovirus, implying that this group of strains may represent
an ancestral clade to classical avian orthoreoviruses.
4 DISCUSSION
Based on the distinguishing biological properties and differences in
the genome structure, WRVs are classified into two groups, novel
and classical WRVs, both belonging to the species Avian orthoreovirus.
Diseases caused by classical and novel WRV infections are character-
ized by similar clinical and pathological signs, but in the latter case,
symptoms tend to be more severe. Weakness, loss of appetite, diar-
rhoea, movement difficulties and lameness are typical clinical signs,
while pathological lesions include hepatitis and splenitis with necrotic
foci, pericarditis and tenosynovitis of the leg tendons (Chen et al.,
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VARGA-KUGLER ET AL . e3389

TA B L E 1 Genomic features of the duck orthoreovirus strain Reo/HUN/DuckDV/2019

Length of the 5’ Protein

Size (bp) end ORF 3’ end Sequence at 5’/3’ end a Encoded protein size (aa)
3998 20 - 3921 - 57 GCUUUU/UUCAUC λA (Core shell) 1306
3899 12 - 3855 - 32 λC (Core turret) 1284
3825 13 - 3780 - 32 λB (Core RdRp) 1259
2282 12 - 2199 - 71 μA (Core NTPase) 732
2150 30 - 2022 - 98 μB (Outer shell) 673
1990 21 - 1908 - 61 μNS (NS factory) 635
22 - 282 - 34 p10 (NS FAST) 93
1573 369 p17 (NS other) 122
1014 σC (Outer fibre) 337
1325 15 - 1251 - 59 σA (Core clamp) 416
1201 30 - 1104 - 67 σB (Outer clamp) 367
1190 23 - 1104 - 63 σNS (NS RNAb) 367
a
Conserved in each segment.

TA B L E 2 Nucleotide (nt) and amino acid (aa) sequence identities (%) of genes between Reo/HUN/DuckDV/2019 and selected avian and
neoavian reoviruses

WRV ARV NeARV Ych, D2533/6

min max min max min max min max

λA nt 70.63% 71.71% 70.96% 72.33% 66.80% 68.23% 94.29% 95.01%
aa 83.40% 84.10% 84.10% 84.79% 75.17% 79.05% 98.14% 99.07%
λB nt 65.52% 66.31% 66.18% 66.84% 62.55% 63.24% 91.92% 94.47%
aa 75.20% 76.15% 74.72% 75.68% 69.48% 69.87% 97.62% 98.25%
λC nt 55.49% 56.16% 54.99% 55.82% 48.35% 48.97% 89.11% 91.30%
aa 55.98% 56.61% 54.26% 55.43% 41.13% 41.20% 95.39% 96.79%
μA nt 57.06% 59.50% 59.22% 59.82% 53.26% 55.28% 92.16% 94.45%
aa 60.33% 61.43% 61.16% 61.98% 53.44% 54.13% 94.21% 98.21%
μB nt 60.67% 64.30% 63.97% 64.69% 65.07% 66.12% 89.77% 94.00%
aa 65.35% 69.97% 67.99% 70.79% 70.96% 74.09% 98.02% 98.18%
μNS nt 57.26% 58.37% 57.79% 58.59% 50.13% 52.53% 93.41% 94.84%
aa 55.82% 57.42% 55.66% 58.05% 41.63% 48.01% 97.29% 97.61%
σA nt 62.14% 63.79% 62.66% 63.71% 55.44% 59.62% 88.86% 94.60%
aa 65.62% 66.67% 65.88% 66.40% 53.81% 59.06% 96.06% 99.21%
σB nt 61.90% 63.24% 56.98% 60.89% 47.37% 48.49% 88.72% 89.05%
aa 57.91% 61.62% 54.88% 59.93% 37.37% 38.05% 98.32% 98.32%
σC nt 38.90% 42.40% 39.57% 41.24% 40.07% 40.23% 86.48% 92.32%
aa 29.65% 31.66% 25.63% 28.64% 26.13% 30.65% 95.48% 97.99%
σNS nt 60.56% 63.03% 60.00% 61.91% 55.17% 57.87% 90.34% 93.93%
aa 61.49% 62.50% 60.81% 63.18% 53.04% 53.72% 96.96% 98.31%

WRV, waterfowl reovirus strains within species Avian orthoreovirus; ARV, chicken and turkey origin reovirus strain (species Avian orthoreovirus); NeARV,
reovirus strains belonging to species Neoavian orthoreovirus; Ich and D2533/6, unusual avian reovirus strains detected in China and Germany, respectively.

2012; Heffels-Redmann et al., 1992; Palya et al., 2003). The strain caused by reoviruses. Common waterfowl viruses associated with
Reo/HUN/DuckDV/2019 was isolated from ducks with decreased egg the observed histopathological lesions could not be detected by PCR
production accompanied by lymphocytic interstitial pneumonia, both screening. Nonetheless, the presence of other co-infecting pathogens
of which are typical of viral infection but less common in infection could not be excluded even if the viral metagenomics approach we
 18651682, 2022, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14654 by Yonsei University Central Library, Wiley Online Library on [25/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
e3390 VARGA-KUGLER ET AL .

F I G U R E 2 Unrooted maximum-likelihood phylogenetic trees based on the nucleotide sequences of the core clamp (σA) and the outer clamp
(σB) protein coding genes. The study strain is marked with a dot. Classical and novel waterfowl origin strains, and unclassified duck origin strains
are indicated with yellow, blue and green colour, respectively. The scale bar is proportional to the genetic distance. The number of bootstrap
replicates was 10,000. Bootstrap values greater than 60 are shown at the branch nodes

utilized on the cytopathogenic isolate of unknown origin failed to
detect any other viruses (data not shown). Similarly, domestic ducks
infected with closely related reovirus strains, D2533/6 and Ych (iso-
lated during the 2010s in Germany and in China, respectively), also
showed some clinical features that are not characteristic to disease
caused by classical/novel WRVs; these include intestinal haemorrhage,
air sacculitis and lymphocyte depletion in the bursa (Cao et al., 2019;
Farkas et al., 2018). These clinical signs and pathological lesions are not
consistent and might be complicated by co-infecting agents possibly
overlooked in these studies.
Novel WRVs that belong to the species Avian orthoreovirus emerged
in the 1990s in China and the associated disease was reported only
from Asia. Classical WRVs were detected in both Europe and Asia, and
the isolates derived from different continents formed phylogenetically
distinct groups based on partial genome sequence data, suggesting
the divergent evolution of WRVs on the two continents (Yun et al.,
2013). On the contrary, whole genome analysis of European and
Asian orthoreovirus isolates clearly demonstrated genetic interaction
between classical and novel WRVs, showing strong evidence of a
series of independent reassortment events in the past between the
two pathotypes of WRVs. Reassortment affected numerous genes,
resulting in complex phylogenetic relationships and an epidemio-
logical pattern of WRVs in domestic ducks and geese (Farkas et al.,
2014, 2018; Wang et al., 2020). However, the μB, σB and σC coding
genome segments whose products are expressed on the surface of
the virion appeared to co-segregate with the pathotypes. It is possible
that the two clades of WRVs (Avian orthoreovirus) co-circulated over
decades between the two continents and bird trading or migration
of wild birds may have played a role in the long-distance dispersal
of these viruses between continents, and the intra-host evolution by
reassortment.
The novel Hungarian orthoreovirus strain,
Reo/HUN/DuckDV/2019, analysed in this study, and strains D2533/6
and Ych were only distantly related to other known avian reoviruses
and neoavian reoviruses. Although the genome structure and the
genome segment termini sequences were similar to those of ARVs
and NeARVs, the pairwise identity data suggested that they might
be classified into a new orthoreovirus species distinct from ARVs.
To distinguish this clade, we propose to use the assignment ‘Avian
orthoreovirus B’. The host spectrum of this proposed new taxon seems,
in part, to overlap with that of species Avian orthoreovirus, but the
true hosts and the origin of isolated strains within candidate ‘Avian
orthoreovirus B’ remain obscure. Waterfowl are traditionally kept in
semi-intensive housing conditions with natural or artificial swimming
facilities where farmed birds readily get in contact with wild birds.
The lack of systemic virus surveillance together with the atypical
pathogenicity could have helped these viruses to remain unnoticed
in domestic waterfowl. There is no commercially available vaccine
for the prevention of WRV infection, thus the detection of novel
waterfowl-associated reoviruses in moribund domestic duck raises
further challenges to disease control and prevention. PCR-based
detection in diagnostic laboratories could readily provide relevant
information concerning the prevalence and disease associations of
emerging strains of the candidate ‘Avian orthoreovirus B’.
ACKNOWLEDGEMENTS
This work was supported by the National Scientific Research Fund of
Hungary (K120201).

VARGA-KUGLER ET AL .
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to. Ethical
approval was not required as no animal experiments were performed.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly avail-
able in GenBank at https://www.ncbi.nlm.nih.gov, reference number
OM908476 to OM908485.
ORCID
Renáta Varga-Kugler https://orcid.org/0000-0002-0093-5418
Krisztián Bányai https://orcid.org/0000-0002-6270-1772
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