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Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations a
a
a
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Katarzyna Domanska-Blicharz , Anna Jacukowicz , Anna Lisowska , Krzysztof Wyrostek a
& Zenon Minta a
Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland Accepted author version posted online: 18 Aug 2014.Published online: 01 Oct 2014.
Click for updates To cite this article: Katarzyna Domanska-Blicharz, Anna Jacukowicz, Anna Lisowska, Krzysztof Wyrostek & Zenon Minta (2014) Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations, Avian Pathology, 43:5, 406-413, DOI: 10.1080/03079457.2014.949619 To link to this article: http://dx.doi.org/10.1080/03079457.2014.949619
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Avian Pathology, 2014 Vol. 43, No. 5, 406–413, http://dx.doi.org/10.1080/03079457.2014.949619
ORIGINAL ARTICLE
Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations Katarzyna Domanska-Blicharz*, Anna Jacukowicz, Anna Lisowska, Krzysztof Wyrostek, and Zenon Minta
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Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland
We examined 884 wild birds mainly from the Anseriformes, Charadriiformes and Galliformes orders for infectious bronchitis (IBV)-like coronavirus in Poland between 2008 and 2011. Coronavirus was detected in 31 (3.5%) of the tested birds, with detection rates of 3.5% in Anseriformes and 2.3% in Charadriiformes and as high as 17.6% in Galliformes. From the 31 positive samples, only 10 gave positive results in molecular tests aimed at various IBV genome fragments: five samples were positive for the RdRp gene, four for gene 3, eight for gene N and eight for the 3′-untranslated region fragment. All analysed genome fragments of the coronavirus strains shared different evolutionary branches, resulting in a different phylogenetic tree topology. Most detected fragment genes seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX). Two waves of coronavirus infections were identified: one in spring (April and May) and another in late autumn (October to December). To our knowledge this is the first report of the detection of different fragment IBV-like genes in wild bird populations.
Introduction Coronaviruses (CoVs) are responsible for a broad spectrum of diseases in a wide variety of animals and humans. Most avian CoVs detected in domestic fowl belong to the Gammacoronavirus genus (order Nidovirales, family Coronaviridae, subfamily Coronavirinae) together with the non-avian CoV SW1 isolated from a beluga whale. Some avian CoVs detected mainly in wild birds, but also mammalian CoVs, are members of the Deltacoronavirus genus (Dong et al., 2007; Carstens, 2010). The virus has a single-stranded, positive-sense RNA genome, approximately 30 kb long, consisting of several open reading frames. Two-thirds of the genome in the 5′ end is occupied by two overlapping open reading frames encoding viral RNA-dependent RNA polymerase (RdRp). At the 3′ end are genes encoding the four major structural proteins— spike (S), envelope (E), matrix (M), and nucleocapsid (N)— with genes 3 and 5 encoding non-structural accessory proteins (Britton et al., 2006; Hodgson et al., 2006). The genome of CoV includes untranslated regions (Papageorgiou et al., 2010) at the 5′ and 3′ genome termini, which play a role in viral RNA synthesis (Sawicki et al., 2007). The main representative of the Gammacoronavirus genus is infectious bronchitis virus (IBV), which is responsible for enormous economic losses in the poultry industry. IBV can cause respiratory and renal diseases in chickens of all ages and also a reduction of egg quality and quantity in mature hens. IBV constantly spreads and affects numerous geographic areas, frequently in new and genetically different
forms that make the virus difficult to control (Worthington et al., 2008). In recent years, avian CoVs have been isolated from various bird orders such as Galliformes, Anseriformes, Columbiformes, Charadriiformes, Passeriformes, Pelacaniformes, Ciconiiformes and Psittaciformes (Cavanagh, 2005; Jonassen et al., 2005; Gough et al., 2006; Qian et al., 2006; Woo et al., 2009; Chen et al., 2010; Chu et al., 2011; Chen et al., 2013). This creates a suspicion that wild birds play a role as CoV reservoirs and as a CoV carrier/transmitter influencing IBV epidemiology. To date only limited studies have been performed to systematically monitor the prevalence of CoVs in wild bird populations and they revealed CoV occurrence in 1.6% and 6.4% of studied samples in northern England and the Beringia area (between Siberia and Alaska), respectively (Hughes et al., 2009; Muradrasoli et al., 2010). Recent wild bird surveillance in Hong Kong and Cambodia demonstrated the presence of CoV in 12.5% of samples studied and a huge diversity in viruses detected (Chu et al., 2011). The main objective of this study was to investigate the presence of IBV-like CoVs in a variety of wild bird species in the territory of Poland and also to perform some preliminary molecular characterization based on selected genome fragments, namely the 3, RdRp and N genes and also the 3′-untranslated region (UTR) fragment, of the avian CoVs detected. Materials and Methods Sample collection. Between January 2008 and December 2011, a total of 884 cloacal/faecal swabs were collected from birds living in areas of high
*To whom correspondence should be addressed: Tel: +48 81 889 30 67. Fax: +48 81 886 25 95. E-mail: [email protected] (Received 15 April 2014; accepted 14 July 2014) © 2014 Houghton Trust Ltd
IBV-like viruses in wild birds
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wild bird concentration in Poland. These samples were originally used for avian influenza virus surveillance purposes. The predominant species of the birds tested was the Anseriformes order and consisted of mallards (292 birds), mute swans (168 birds) and birds described as “goose” (140 birds). The second largest bird representation in the study was Charadriiformes (106 birds) and consisted of different species of gulls (Table 1). The remaining samples (73 birds) came from other birds, such as pheasants, Eurasian coots, common cranes, quails, white storks, and so forth. Each individual swab was hydrated in phosphate-buffered saline supplemented with antibiotics (100 u penicillin and 100 mg streptomycin/ml), incubated for 1 h at room temperature and clarified by centrifugation at 1500 × g for 20 min. If the swabs originated from the same species and place, they were pooled (up to 5 swabs/pool) and used for RNA extraction. Positive samples were re-examined using individual swabs. To investigate whether any relationship existed between the presence of CoV and the time (month) of sampling, the data collected were tested using tools available in Excel (Microsoft Office Excel, 2007). Molecular methods and phylogeny. Total RNA was extracted from 250 µl individual or pooled supernatants using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Each RNA was eluted in 50 µl RNase-free water and then checked for the presence of IBV-like CoV using the method that copies the conserved region of the 5′UTR in real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) according to Callison et al. (2006). This method is routinely used in our laboratory for IBV and turkey coronavirus (TCoV) identification. The rRT-PCR was performed using the QiantiTect Probe RT-PCR Kit (QIAGEN) according to the manufacturer’s instructions using the ABI 7300 real-time PCR machine (Applied Biosystems Inc., Foster City, CA, USA). The 5′UTR-positive samples were then examined using different RT-PCRs: for the 3 gene and the 3′UTR fragment, amplification with combinations of the primers described by Cavanagh et al. (2002); and for the RdRp and N genes, amplifications with primers according to Maurel et al. (2011). In all reactions, the 4/91 vaccine strain (793B-like group of IBVs) was used as positive control. The products were separated on a 2% agarose gel in Trisacetate–ethylenediamine tetraacetic acid buffer and visualized using ethidium bromide stain. Amplicons were purified using a NucleoSpin Extract II Kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s instructions and sequenced at the commercial service (Genomed, Warsaw, Poland). Using the SeqManPro program (DNASTAR Inc., Madison, WI, USA), the forward and reverse nucleotide sequences were aligned as one consensus sequence. The sequences were analysed with MEGA 5.0 (Tamura et al. 2011) using the neighbour-joining method with the maximum-likelihood model. Bootstrap scores were generated from 1000 replicates. Identities between aligned sequences were calculated with the MEGA 5.0 program. Accession numbers. The gene sequences were submitted to the GenBank database and have been assigned the following accession numbers: the partial RdRp gene sequences of ACoV/Duck/PL-MW283/2009, ACoV/ Duck/PL-MW284/2009, ACoV/Mallard/PL-MW345/2009, ACoV/Bean goose/PL-MW434/2009 and ACoV/Bean goose/PL-MW435/2009 are KJ690951 to KJ690955; the partial 3 gene sequences of ACoV/Mallard/ PL-MW150/09, ACoV/Goose/PL-MW162/2009, ACoV/Duck/PL-MW283/ 2009 and ACoV/Duck/PL-MW284/2009 are KJ690947 to KJ690950; and the partial N gene sequences of ACoV/Goose/PL-MW162/2009, ACoV/Mallard/PL-MW272/2011, ACoV/Duck/PL-MW284/2009, ACoV/ Duck/PL-MW371/2009, ACoV/Bean goose/PL-MW434/2009, ACoV/Bean
Table 1.
407
goose/PL-MW435/2009, ACoV/Mallard/PL-MW123/2010 and ACoV/Duck/ PL-MW283/2009 are KJ690956 to KJ690963, respectively. The accession numbers for the 3′UTR fragment sequences of ACoV/Goose/PL-MW162/2009, ACoV/Duck/PL-MW283/2009, ACoV/Duck/PLMW284/2009, ACoV/Mallard/PL-MW345/2009, ACoV/Duck/PL-MW371/2009, ACoV/Bean goose/ MW434/2009, ACoV/Bean goose/PL-MW435/2009 and ACoV/Mallard/PLMW272/2011 are KJ690964 to KJ690971, respectively.
Results Surveillance study. Out of the 884 wild birds tested, 31 were positive (3.5%) (Table 2): one bird (3.3%) tested in 2008, 13 birds (3.8%) tested in 2009, eight birds (2.5%) tested in 2010 and nine birds (4.9%) tested in 2011. They originated from different species of Anseriformes—ducks (17 birds including 12 mallards), geese (six birds) and mute swans (two birds)—but also from Charadriiformes (three gulls) and Galliformes (three pheasants). Our analyses indicate the presence of CoVs in samples collected from the wild birds in the following months: April, 9/55 (16.4%); May, 8/100 (8%); October, 9/267 (3.4%); November, 2/182 (1.1%); and December, 2/55 (3.6%). Molecular analysis. All 31 positive samples were subjected to RT-PCR aimed at amplification of other CoV gene fragments. Only 10 of them gave positive results (Table 3). Five samples were positive for the RdRp gene, four for gene 3, eight for gene N, and eight for the 3′UTR fragment. In this study, all positive amplicons were sequenced. Each analysed CoV-strain gene shared a different evolutionary branch that resulted in a different phylogenetic tree topology (Figures 1 to 4). With reference to the RdRp gene, about 680-nucleotide-long sequences of the five ACoVpositive wild birds were 88.1 to 100% homologous to each other and clustered in two groups closely related to IBV and TCoV strains and were distantly related to recently described CoVs found in wild birds from the Beringia area (Figure 1). Three strains from Anatinae birds (ACoV/ Mallard/PL-MW345/2009, ACoV/Duck/PL-MW283/2009 and ACoV/Duck/PL-MW284/2009) formed a common cluster and their nucleotide homology was between 92.7 and 100%. Two strains from Anserinae birds (ACoV/ Bean goose/PLMW434/2009 and ACoV/Bean goose/PLMW435/2009) formed a separate cluster distantly located from the IBV/TCoV strains. It was noteworthy that the strains which had identical RdRp gene sequences were isolated at the same sampling site and at the same time. In relation to TCoV and IBV the nucleotide identity was between 88.1 and 95.4%, and in relation to CoVs of the wild birds from the Beringia area it was 73.4 to 81.6%. Sequence analysis of 840 nucleotides of gene 3 showed that all four CoV from wild birds had close similarities
Overview of wild bird samples examined in this study.
Species of bird Year
Number of samples
Swans
Mallards
Ducks
Geese
Gulls
Other wild birds
2008 2009 2010 2011 Total
30 346 323 185 884
10 64 65 29 168
4 117 102 69 292
0 47 33 25 105
7 37 60 36 140
0 55 28 23 106
9 26 35 3 73
408
K. Domanska-Blicharz et al. Table 2.
Order
Group
Species
Number sampled
Number positive
Percent positive
Anseriformes
Swans
Cygnus olor Cygus cygnus Anser albifrons Anser anser Anser fabalis undefined Anas platyrhynchos Anas querquedula Anas crecca Branta canadensis undefined Gallinago gallinago Larus ridibundus Larus canus Larus argentatus Sterna hirundo Sturnus vulgaris Columba livia Grus grus Fulica atra Phasianus colchicus Coturnix coturnix Perdix perdix Lyrurus tetrix Ciconia ciconia Falco tinnunculus Accipiter gentilis Aquila chrysaetos Dromaius novaehollandiae Ardea cinerea 28 defined
161 7 27 33 30 50 292 20 30 2 53 14 43 57 6 7 1 6 4 10 10 4 2 1 2 3 2 1 2 4 884
2 0 0 1 0 5 12 1 0 0 4 0 3 0 0 0
1.2 –a – 3.0 – 10.0 4.1 5.0 – – 7.5 – 7.0 – – – – – – – 30.0 – – – – – – – – – 3.5
Geese
Ducks
Charadriiformes
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Overview of the prevalence of avian coronaviruses in the samples studied.
Waders Gulls
Tern Passeriformes Columbiformes Gruiformes
Pigeon
Galliformes
Ciconiiformes Falconiformes Acciptriformes Casuariiformes Pelecaniformes Total: 11 a
0 0 0 3 0 0 0 0 0 0 0 0 0 31
No positive samples.
with each other, ranging from 90.0 to 99.6%, and were different from IBV and TCoV, with identities ranging from 85.2 to 88.3%. They formed a separate cluster with the bootstrap value of 95% (Figure 2). Three strains of ACoVs (ACoV/Duck/PL-MW283/2009, ACoV/Duck/PL-MW284/ 2009 and ACoV/Goose/PL-MW162/2009) were 98.1 to 99.6% similar to each other and they originated from the birds caught at different times but at the same ornithological
observation site (Dolnoslaskie). On the other hand, the gene 3 sequence of ACoV/Mallard/PL-MW150/2009 diverged from the other, with a nucleotide identity of 90.0 to 90.7%; this originated from the birds caught about 500 km away from Dolnoslaskie. With regard to the N gene (about 840 nucleotide), eight of the analysed fragment CoV strains fell into two different groups in the phylogenetic tree and the nucleotide
Table 3. Details of coronaviruses from wild bird samples that were molecularly characterized in this study.
Strain of avian CoV (species/unique number/year) 1 2 3 4 5 6 7 8 9 10 a
Mallard/PL-MW150/2009 Goose/PL-MW162/2009 Duck/PL-MW283/2009 Duck/PL-MW284/2009 Mallard/PL-MW345/2009 Duck/PL-MW371/2009 Bean goose/PLMW434/2009 Bean goose/PLMW435/2009 Mallard/PL-MW123/2010 Mallard/PL-MW272/2011
N gene
3′ UTR
+ + + + – – –
– + + + – + +
– + + + + + +
+
–
+
+
– –
– –
+ +
– +
Place/part of Poland
Sample date
Zelwa lake/north eastern Bukowka reservoir/south western Bukowka reservoir/south western Bukowka reservoir/south western Stary Lubliniec reservoir/south eastern Bukowka reservoir/south western Bukowka reservoir/south western
29 April 6 May 2 October 2 October 23 October 4 November 16 December
–a – + + + – +
Bukowka reservoir/south western
16 December
Jeziorsko reservoir/central Zywieckie lake/southern
26 April 26 October
Negative result in respective RT-PCR protocol.
RdRp gene 3 gene
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IBV/Conn46 1983 (FJ904718) TCoV/TX-GL/01 (GQ427174) IBV/Cal557/2003 (FJ904715) IBV/Ark DPI (GQ504720) IBV/Georgia 1998 8p(GQ504722) ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 IBV/Mass41 Vaccine (GQ504725) ACoV/Peafowl/GD/KQ6/2003 (AY641576) IBV/CH/LDL/101212 (JF828981) IBV/H120 (GU393335) TCoV/Inn-517/94 (GQ427175) TCoV ATCC (EU022526) IBV/ITA/90254/2005 (FN430414) TCoV/FR080147C (FN811145) TCoV/FR070341J (FN811144) TCoV/FR080183J (FN811146)
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ACoV/Mallard/PL-MW345/2009 ACoV/Quail/Italy/Elvia/2005 (EF446156) IBV/vaccine strain 4/91 (KF377577) ACoV/Duck/CH/HN/ZZ2004 (JF705860) IBV/SAIBK (DQ288927) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Pintail/PBA-10 (GU396668) ACoV/Pintail/PBA-124 (GU396673 ) ACoV/Glaucous-winged gull/CIR-66002 (GU396682) ACoV/Rock sandpiper/CIR-65824 (GU396689) ACoV/Rock sandpiper/CIR-65828 (GU396689) ACoV/Brent goose/KR69 (GU396677) ACoV/Brent goose/KR70 (GU396676) Beluga whale CoV/SW1 (EU111742) ACoV/Anas americana/100112 (JN788849) ACoV/Ardea cinerea/091223 (JN788874) ACoV/Anas crecca/091230 (JN788837) ACoV/Platalea minor/091127 (JN788788) ACoV/Trush/HKU12-600 (FJ376621) ACoV/Common-moorhen/HKU21-8295 (JQ065049) ACoV/White-eye/HKU16-6847 (JQ065044) Asian leopard-cat CoV (EF584908) ACoV/Magpie-robin/HKU18-chu3 (JQ065046) ACoV/Munia/HKU13-3514 (FJ376622)
0.05
Figure 1. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the RdRp gene of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura twoparameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
homology between them was 89.9 to 100% (Figure 3). Seven of these were in the “793B-like” cluster, with a significance of 63% for the bootstrap value. Similarly to the RdRp gene, the N genes of Anatinae and Anserinae strains isolated at the same sampling site and at the same time were the most closely related and formed two distinct clusters with nucleotide homology between them of 95.2%. The strains ACoV/Goose/PL-MW162/2009 and ACoV/Duck/ PL-MW371/2009 were most closely related to the 793Blike 4/91 IBV strain (95.5 to 95.8%), the component of the second most frequently used vaccine in Poland. The eighth strain, ACoV/Mallard/PL-MW123/2010, was in the “QXlike” cluster together with strains from Italy and Sweden
despite their nucleotide homology being between 91.5 and 93.7%. The 3′UTR fragment sequences (approximately 266 nucleotides) of eight CoVs detected in wild birds in Poland clustered into two groups and the nucleotide homology between them was 98.8 to 100% (Figure 4). Three ACoVs (ACoV/Bean goose/PL-MW435/2009, ACoV/Bean goose/ PL-MW434/2009 and ACoV/Mallard/PL-MW345/2009) shared 100% nucleotide sequence identity to each other and with the sequence of the North American IBV/DE072 strain and the Chinese IBV/ck/CH/LJC/111,054 strain. Five additional CoV strains that gave positive 3′UTR amplicons had nucleotide homology between 99.6 and 100% and
410
K. Domanska-Blicharz et al. IBV/ITA/90254/2005 (FN430414) IBV/SWE/0658946/10 (JQ088078) ACoV/Partridge/GD/S14/2003 (AY646283) IBV/CK/CH/LLN/98I (EF602451) ACoV/Duck/DK/CH/HN/2004 (HM371091) IBV/SAIBK (DQ288927) ACoV/Mallard/PL-MW150/2009 ACoV/Goose/PL-MW162/2009 ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 IBV/V2-71 (DQ490216) IBV/vaccine strain 4/91 (KF377577) IBV/Georgia 1998 pass8 (GQ504722) IBV/Delaware 072 (GU393332) IBV/Arkansas Vaccine (GQ504721) IBV/Conn46 1996 (FJ904716) IBV/CK/CH/LTJ/95I (EF602448)
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TCoV/TX-GL/01 (GQ427174) IBV/Massachusetts (GQ504724) ACoV/Peafowl/GD/KQ6/2003 (AY641576) IBV/Beaudette CK (AJ311317) IBV/CH/LDL/101212 (JF828981) IBV/H120 (GU393335) Beluga whale CoV/SW1 (EU111742) ACoV/Common-moorhen/HKU21-8295 (JQ065049) ACoV/White-eye/HKU16-6847 (JQ065044) ACoV/Thrush/HKU12-600 (FJ376621) ACoV/Munia/HKU13-3514 (FJ376622) ACoV/Magpie-robin/HKU18-chu3 (JQ065046) ACoV/Sparrow/HKU17-6124 (JQ065045)
0.1
Figure 2. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of gene 3 of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura two-parameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
grouped together with the 4/91 vaccine strain. Divergence at the nucleotide level between these two groups was 1.5%.
Discussion In the present study, the vast majority of birds tested belonged to Anseriformes (79.7%), and Anas platyrhynchos and Cygnus olor constituted 33% and 18.2% of all birds tested, respectively. The next largest group of bird orders represented were Charadriiformes and Galliformes, which made up 14.4% and 2% of birds tested, respectively. During the 4-year period, the presence of coronaviral RNA was detected in 3.5% of all wild birds examined, and in samples originating from the most represented bird orders. The detection rate was 3.5% in Anseriformes and 2.3% in Charadriiformes, but as high as 17.6% in Galliformes even though this bird order was the lowest represented. In previous studies, the average prevalence in wild birds was 1.6% in northern England, over 6% in the Bering Strait Area (Beringia) and 12.5% in Hong Kong and Cambodia (Hughes et al., 2009; Muradrasoli et al., 2010; Chu et al., 2011). However, these results could not reflect the real prevalence of CoVs due to the fact that all studies have used different molecular methods for CoV detection and also due
to the huge diversity of CoVs, meaning that some of them could be missed. Recently, the application of the pancoronavirus molecular method aimed at the RdRp sequence has allowed the identification of a new genus of avian CoV, Deltacoronavirus (Woo et al., 2009; Chu et al., 2011). These viruses were detected mainly in Ciconiiformes, Pelecaniformes, Charadriiformes and Passeriformes (superorder Neoaves) and also, but to a lesser extent, in Anseriformes (superorder Galloanserae). In contrast, the occurrence of gammacoronaviruses was reported predominantly in Galloanserae. The authors of this survey concluded that deltacoronaviruses could have a more stringent host specificity than gammacoronaviruses, among which frequent intergenus and interspecies transmission is observed (Chu et al., 2011). The method used in our study enabled the detection of gammacoronavirus of poultry (IBV and TCoV), so it focused on the detection of similar viruses in wild birds. Because of this, the demonstration of the highest prevalence of CoVs in wild birds from the Galliformes order is not surprising. Other CoVs were detected in wild waterfowl of the orders Anseriformes and Charadriiformes such as swans, graylag geese, mallards, garganeys and black-headed gulls, but the detection rate in these orders was lower. A recent Chinese survey of domestic fowl
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ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 ACoV/Duck/PL-MW371/2009 ACoV/Goose/PL-MW162/2009 IBV/vaccine strain 4/91 (KF377577) TCoV/France/ FR080147c (FN665665) TCoV/France/FR080183j (FN665666) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Mallard/PL-MW272/2011 IBV/ITA90254/2005 (FN430414) ACoV/Mallard/PL-MW123/2010 IBV SWE/0658946/10 (JQ088078) IBV/CH/LDL/101212 (JF828981 ) IBV/H120 (GU393335) IBV/Cal557/2003 (FJ904715 ) IBV/Georgia 1998 (GQ504722)
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IBV H52 (EU817497) ACoV/Peafowl/GD/KQ6/2003 (AY641576) TCoV ATCC (EU022526) IBV/ArkDPI (FJ904719) IBV/Conn46/1983 ((FJ904718) TCoV/In-517/94 (GQ427175) TCoV/TX-GL-01 (GQ427174) IBV/SAIBK (DQ288927) Beluga whale CoV/SW1 (EU111742) ACoV/Bulbul/HKU11-934 (FJ376619) ACoV/Thrush/HKU12-600 (FJ376621) ACoV/Munia/HKU13-3514 (FJ376622) Asian leopard cat CoV/Guangxi/F230/2006 (EF584908)
0.1
Figure 3. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the N gene of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura two-parameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
revealed the presence of the N gene of IBV-like viruses in 4.25% of chickens but also in 0.80% of ducks and 1.82% of geese. These CoVs grouped together with IBV from chickens in different clades of the phylogenetic tree, resulting from the analysis of N gene sequences (Chen et al., 2013). The observed differences in avian CoV prevalence in wild birds could also result from the geographical location of sampling and/or the years of research. Results of Norwegian studies revealed that the percentage of CoVpositive birds in 2004 was 38%, and in 2003 was only 16% (Jonassen et al., 2005). In our study, the yearly CoV prevalence was similar and fluctuated around 3.5%, with the lowest value of 2.5% in 2010 and the highest value of 4.9% in 2011. Additionally, similar to avian influenza virus, the frequency of CoV detection may also be connected with the season. We investigated the seasonal pattern of CoV circulation in wild birds during the whole year, and two waves of infection were found. The first wave, between 3.4 and 3.6%, occurred in late autumn (October to December) and this pattern is similar to avian influenza virus circulation in wild birds (Munster et al., 2007). All 14 CoVinfected birds in this season belonged to Anseriformes. This wave of CoV prevalence may be connected with autumn migration when young and immunologically naive birds could be carrying CoV or become infected with local
circulating CoV. The second wave of CoV prevalence, with a rate of 8 to 16.4%, was observed in late spring (April to May). Among 17 CoV-infected birds, six belonged to Charadriiformes and Galliformes and 11 to Anseriformes. This epidemiological cycle of CoV coincides with the breeding season and may be the result of the increased need for food, and also possible contact with domestic poultry. Most positive wild birds originated from the areas surrounding artificial water retention, such as the Bukówka reservoir situated in the valley of the river Bóbr. Many poultry farms are located in this geographic region. There are also a few villages where backyard poultry are held, thus creating favourable conditions for contact with wild birds. No information was collected regarding clinical signs or body parameters in the CoV-infected birds. We therefore reached no conclusion as to whether these infections had any influence on health status. Previous reports are ambiguous, as numerous CoV-positive wild birds were described as healthy (Jonassen et al., 2005; Liu et al., 2005; Sun et al., 2007; Hughes et al., 2009). However, CoV infection in wild birds reported in other papers had a clinical significance for the birds (Jonassen et al., 2005; Gough et al., 2006; Qian et al., 2006; Circella et al., 2007). Out of 31 positive results in the rRT-PCR specific for the 5′UTR genome fragment, only 10 gave positive results in
412
K. Domanska-Blicharz et al. ACoV/Anas/p42/2005/GBR (FJ490199) ACoV/Oystercatcher/p17/2006/GBR (FJ490198) ACoV/Red knot/p60/2006/GBR (FJ490197) TCoV-ATCC (EU022526) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Mallard/PL-MW345/2009 IBV/DE072 (AF203002) TCoV/TX-1038/98 (GQ427176) IBV/NGA/A116E7/2006 (FN430415) IBV/Beaudette US (AJ311362) IBV/ArkDPI11 (EU418976) IBV partridge/GD/S14/2003 (AY646283) ACoV/Partridge/GD/S14/2003 (AY646283) IBV/vaccine strain 4/91 (KF377577) IBV/ITA/90254/2005 (FN430414) IBV/H120 (GU393335)
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IBV TCoV/TX-1038/98 (GQ427176) IBV CK/SWE/0658946/10 (JQ088078) ACoV/Mallard/PL-MW272/2011 ACoV/Goose/PL-MW162/2009 ACoV/Anas/UK/p33/2005 (FJ490195) ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 ACoV/Duck/PL-MW371/2009 ACoV/Whooper swan/UK/p3/2005 (FJ490193) IBV/strain Mass41 (GQ504725) ACoV/Peafowl/GD/KQ6/2003 (AY641576) Beluga whale CoV/SW1 (|EU111742) ACoV/Thrush CoV/HKU12-600 (FJ376621) ACoV/Common-moorhen CoV/HKU21-8295 (JQ065049) ACoV/Magpie-robin CoV/HKU18-chu3 (JQ065046) ACoV/White-eye CoV/HKU16-6847 (JQ065044)
0.1
Figure 4. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the 3′UTR fragment of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using neighbour-joining method and the Kimura twoparameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
RT-PCRs aimed at other fragments of the IBV-like genome. To our knowledge, this is the first report of the analysis of different fragments of the IBV-like genome detected in wild bird populations. Most of the studies on the prevalence and molecular characterization of CoV in wild birds were based on the detection of just one genome fragment. Usually this was the RdRp gene, but others such as the N gene or the 3′UTR fragment have also been investigated (Jonassen et al., 2005; Hughes et al., 2009; Muradrasoli et al., 2010). In our study, we focused on four IBV genome fragments—RdRp, the 3 gene, the N gene and the 3′UTR fragment—with most positive amplicons being obtained in PCRs with primers directed to the N gene and 3′UTR fragment, and the least positives in the case of PCRs aimed at genes 3 and RdRp. The reason for failures in other RTPCR attempts can include either a very high sensitivity of the rRT-PCR method for CoV detection or, more probably, the gene sequence variability of the CoV detected, so that the primers used did not match them. The four phylogenetic trees shown here, based on the RdRp, genes 3 and N, and the 3′UTR, show that viruses from wild birds segregated with other avian CoVs,
including IBV, TCoV and viruses from quail, peafowl and duck, indicating that these viruses are IBV-like gammacoronaviruses. However, for each analysed genome fragment, CoV strains from wild birds shared a different evolutionary branch that resulted in a different location on the phylogenetic trees. Most gene fragments detected seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX) (Domanska-Blicharz et al., 2006, 2007). CoVs detected in northern England originating from birds of the Anseriformes and Charadriiformes orders had genomic 3′UTR fragments phylogenetically related to IBV. Moreover, virus sequences from most CoV samples shared high homology with the IBV H120 vaccine strain. Only a few of these were divergent, and more clustered with the 3′UTR sequence of a North American IBV and a TCoV. CoVs similar to IBV H120 vaccine strain were also described in healthy, unvaccinated, domestic peafowl and wild peafowl in China, where their presence probably arose from widespread use of IBV vaccines in the local poultry population (Liu et al., 2005). The 3′UTR fragment of most of the CoVs analysed in this study also shared high
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nucleotide identity with the sequence of the IBV H120 vaccine strain, commonly used for the immunoprotection of commercial chickens in Poland. Interestingly, three of them had high nucleotide similarity to a previously identified and unusual CoV detected in northern England (Hughes et al., 2009). In conclusion, our results described the prevalence of CoVs in the wild bird population in Poland. The viruses detected are IBV-like gammacoronaviruses, which carry selected gene fragments of different lineages of IBV. The source of wild bird infections with IBV-like CoV was probably poultry, and IBV genome detection could suggest that wild birds might constitute the mobile genome deposit of the CoV carrying gene pool, even in a huge geographic territory. Also, the possibility of recombination between different CoV strains, when they infect the same wild bird host, could constitute the mixing vessel where new variants of CoV arise. Acknowledgements The authors would like to thank Lukasz Bocian from the Department of Epidemiology and Risk Assessment, National Veterinary Research Institute for his help with analysis of the data obtained.
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Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations a
a
a
a
Katarzyna Domanska-Blicharz , Anna Jacukowicz , Anna Lisowska , Krzysztof Wyrostek a
& Zenon Minta a
Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland Accepted author version posted online: 18 Aug 2014.Published online: 01 Oct 2014.
Click for updates To cite this article: Katarzyna Domanska-Blicharz, Anna Jacukowicz, Anna Lisowska, Krzysztof Wyrostek & Zenon Minta (2014) Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations, Avian Pathology, 43:5, 406-413, DOI: 10.1080/03079457.2014.949619 To link to this article: http://dx.doi.org/10.1080/03079457.2014.949619
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Avian Pathology, 2014 Vol. 43, No. 5, 406–413, http://dx.doi.org/10.1080/03079457.2014.949619
ORIGINAL ARTICLE
Detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations Katarzyna Domanska-Blicharz*, Anna Jacukowicz, Anna Lisowska, Krzysztof Wyrostek, and Zenon Minta
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Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland
We examined 884 wild birds mainly from the Anseriformes, Charadriiformes and Galliformes orders for infectious bronchitis (IBV)-like coronavirus in Poland between 2008 and 2011. Coronavirus was detected in 31 (3.5%) of the tested birds, with detection rates of 3.5% in Anseriformes and 2.3% in Charadriiformes and as high as 17.6% in Galliformes. From the 31 positive samples, only 10 gave positive results in molecular tests aimed at various IBV genome fragments: five samples were positive for the RdRp gene, four for gene 3, eight for gene N and eight for the 3′-untranslated region fragment. All analysed genome fragments of the coronavirus strains shared different evolutionary branches, resulting in a different phylogenetic tree topology. Most detected fragment genes seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX). Two waves of coronavirus infections were identified: one in spring (April and May) and another in late autumn (October to December). To our knowledge this is the first report of the detection of different fragment IBV-like genes in wild bird populations.
Introduction Coronaviruses (CoVs) are responsible for a broad spectrum of diseases in a wide variety of animals and humans. Most avian CoVs detected in domestic fowl belong to the Gammacoronavirus genus (order Nidovirales, family Coronaviridae, subfamily Coronavirinae) together with the non-avian CoV SW1 isolated from a beluga whale. Some avian CoVs detected mainly in wild birds, but also mammalian CoVs, are members of the Deltacoronavirus genus (Dong et al., 2007; Carstens, 2010). The virus has a single-stranded, positive-sense RNA genome, approximately 30 kb long, consisting of several open reading frames. Two-thirds of the genome in the 5′ end is occupied by two overlapping open reading frames encoding viral RNA-dependent RNA polymerase (RdRp). At the 3′ end are genes encoding the four major structural proteins— spike (S), envelope (E), matrix (M), and nucleocapsid (N)— with genes 3 and 5 encoding non-structural accessory proteins (Britton et al., 2006; Hodgson et al., 2006). The genome of CoV includes untranslated regions (Papageorgiou et al., 2010) at the 5′ and 3′ genome termini, which play a role in viral RNA synthesis (Sawicki et al., 2007). The main representative of the Gammacoronavirus genus is infectious bronchitis virus (IBV), which is responsible for enormous economic losses in the poultry industry. IBV can cause respiratory and renal diseases in chickens of all ages and also a reduction of egg quality and quantity in mature hens. IBV constantly spreads and affects numerous geographic areas, frequently in new and genetically different
forms that make the virus difficult to control (Worthington et al., 2008). In recent years, avian CoVs have been isolated from various bird orders such as Galliformes, Anseriformes, Columbiformes, Charadriiformes, Passeriformes, Pelacaniformes, Ciconiiformes and Psittaciformes (Cavanagh, 2005; Jonassen et al., 2005; Gough et al., 2006; Qian et al., 2006; Woo et al., 2009; Chen et al., 2010; Chu et al., 2011; Chen et al., 2013). This creates a suspicion that wild birds play a role as CoV reservoirs and as a CoV carrier/transmitter influencing IBV epidemiology. To date only limited studies have been performed to systematically monitor the prevalence of CoVs in wild bird populations and they revealed CoV occurrence in 1.6% and 6.4% of studied samples in northern England and the Beringia area (between Siberia and Alaska), respectively (Hughes et al., 2009; Muradrasoli et al., 2010). Recent wild bird surveillance in Hong Kong and Cambodia demonstrated the presence of CoV in 12.5% of samples studied and a huge diversity in viruses detected (Chu et al., 2011). The main objective of this study was to investigate the presence of IBV-like CoVs in a variety of wild bird species in the territory of Poland and also to perform some preliminary molecular characterization based on selected genome fragments, namely the 3, RdRp and N genes and also the 3′-untranslated region (UTR) fragment, of the avian CoVs detected. Materials and Methods Sample collection. Between January 2008 and December 2011, a total of 884 cloacal/faecal swabs were collected from birds living in areas of high
*To whom correspondence should be addressed: Tel: +48 81 889 30 67. Fax: +48 81 886 25 95. E-mail: [email protected] (Received 15 April 2014; accepted 14 July 2014) © 2014 Houghton Trust Ltd
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wild bird concentration in Poland. These samples were originally used for avian influenza virus surveillance purposes. The predominant species of the birds tested was the Anseriformes order and consisted of mallards (292 birds), mute swans (168 birds) and birds described as “goose” (140 birds). The second largest bird representation in the study was Charadriiformes (106 birds) and consisted of different species of gulls (Table 1). The remaining samples (73 birds) came from other birds, such as pheasants, Eurasian coots, common cranes, quails, white storks, and so forth. Each individual swab was hydrated in phosphate-buffered saline supplemented with antibiotics (100 u penicillin and 100 mg streptomycin/ml), incubated for 1 h at room temperature and clarified by centrifugation at 1500 × g for 20 min. If the swabs originated from the same species and place, they were pooled (up to 5 swabs/pool) and used for RNA extraction. Positive samples were re-examined using individual swabs. To investigate whether any relationship existed between the presence of CoV and the time (month) of sampling, the data collected were tested using tools available in Excel (Microsoft Office Excel, 2007). Molecular methods and phylogeny. Total RNA was extracted from 250 µl individual or pooled supernatants using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Each RNA was eluted in 50 µl RNase-free water and then checked for the presence of IBV-like CoV using the method that copies the conserved region of the 5′UTR in real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) according to Callison et al. (2006). This method is routinely used in our laboratory for IBV and turkey coronavirus (TCoV) identification. The rRT-PCR was performed using the QiantiTect Probe RT-PCR Kit (QIAGEN) according to the manufacturer’s instructions using the ABI 7300 real-time PCR machine (Applied Biosystems Inc., Foster City, CA, USA). The 5′UTR-positive samples were then examined using different RT-PCRs: for the 3 gene and the 3′UTR fragment, amplification with combinations of the primers described by Cavanagh et al. (2002); and for the RdRp and N genes, amplifications with primers according to Maurel et al. (2011). In all reactions, the 4/91 vaccine strain (793B-like group of IBVs) was used as positive control. The products were separated on a 2% agarose gel in Trisacetate–ethylenediamine tetraacetic acid buffer and visualized using ethidium bromide stain. Amplicons were purified using a NucleoSpin Extract II Kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s instructions and sequenced at the commercial service (Genomed, Warsaw, Poland). Using the SeqManPro program (DNASTAR Inc., Madison, WI, USA), the forward and reverse nucleotide sequences were aligned as one consensus sequence. The sequences were analysed with MEGA 5.0 (Tamura et al. 2011) using the neighbour-joining method with the maximum-likelihood model. Bootstrap scores were generated from 1000 replicates. Identities between aligned sequences were calculated with the MEGA 5.0 program. Accession numbers. The gene sequences were submitted to the GenBank database and have been assigned the following accession numbers: the partial RdRp gene sequences of ACoV/Duck/PL-MW283/2009, ACoV/ Duck/PL-MW284/2009, ACoV/Mallard/PL-MW345/2009, ACoV/Bean goose/PL-MW434/2009 and ACoV/Bean goose/PL-MW435/2009 are KJ690951 to KJ690955; the partial 3 gene sequences of ACoV/Mallard/ PL-MW150/09, ACoV/Goose/PL-MW162/2009, ACoV/Duck/PL-MW283/ 2009 and ACoV/Duck/PL-MW284/2009 are KJ690947 to KJ690950; and the partial N gene sequences of ACoV/Goose/PL-MW162/2009, ACoV/Mallard/PL-MW272/2011, ACoV/Duck/PL-MW284/2009, ACoV/ Duck/PL-MW371/2009, ACoV/Bean goose/PL-MW434/2009, ACoV/Bean
Table 1.
407
goose/PL-MW435/2009, ACoV/Mallard/PL-MW123/2010 and ACoV/Duck/ PL-MW283/2009 are KJ690956 to KJ690963, respectively. The accession numbers for the 3′UTR fragment sequences of ACoV/Goose/PL-MW162/2009, ACoV/Duck/PL-MW283/2009, ACoV/Duck/PLMW284/2009, ACoV/Mallard/PL-MW345/2009, ACoV/Duck/PL-MW371/2009, ACoV/Bean goose/ MW434/2009, ACoV/Bean goose/PL-MW435/2009 and ACoV/Mallard/PLMW272/2011 are KJ690964 to KJ690971, respectively.
Results Surveillance study. Out of the 884 wild birds tested, 31 were positive (3.5%) (Table 2): one bird (3.3%) tested in 2008, 13 birds (3.8%) tested in 2009, eight birds (2.5%) tested in 2010 and nine birds (4.9%) tested in 2011. They originated from different species of Anseriformes—ducks (17 birds including 12 mallards), geese (six birds) and mute swans (two birds)—but also from Charadriiformes (three gulls) and Galliformes (three pheasants). Our analyses indicate the presence of CoVs in samples collected from the wild birds in the following months: April, 9/55 (16.4%); May, 8/100 (8%); October, 9/267 (3.4%); November, 2/182 (1.1%); and December, 2/55 (3.6%). Molecular analysis. All 31 positive samples were subjected to RT-PCR aimed at amplification of other CoV gene fragments. Only 10 of them gave positive results (Table 3). Five samples were positive for the RdRp gene, four for gene 3, eight for gene N, and eight for the 3′UTR fragment. In this study, all positive amplicons were sequenced. Each analysed CoV-strain gene shared a different evolutionary branch that resulted in a different phylogenetic tree topology (Figures 1 to 4). With reference to the RdRp gene, about 680-nucleotide-long sequences of the five ACoVpositive wild birds were 88.1 to 100% homologous to each other and clustered in two groups closely related to IBV and TCoV strains and were distantly related to recently described CoVs found in wild birds from the Beringia area (Figure 1). Three strains from Anatinae birds (ACoV/ Mallard/PL-MW345/2009, ACoV/Duck/PL-MW283/2009 and ACoV/Duck/PL-MW284/2009) formed a common cluster and their nucleotide homology was between 92.7 and 100%. Two strains from Anserinae birds (ACoV/ Bean goose/PLMW434/2009 and ACoV/Bean goose/PLMW435/2009) formed a separate cluster distantly located from the IBV/TCoV strains. It was noteworthy that the strains which had identical RdRp gene sequences were isolated at the same sampling site and at the same time. In relation to TCoV and IBV the nucleotide identity was between 88.1 and 95.4%, and in relation to CoVs of the wild birds from the Beringia area it was 73.4 to 81.6%. Sequence analysis of 840 nucleotides of gene 3 showed that all four CoV from wild birds had close similarities
Overview of wild bird samples examined in this study.
Species of bird Year
Number of samples
Swans
Mallards
Ducks
Geese
Gulls
Other wild birds
2008 2009 2010 2011 Total
30 346 323 185 884
10 64 65 29 168
4 117 102 69 292
0 47 33 25 105
7 37 60 36 140
0 55 28 23 106
9 26 35 3 73
408
K. Domanska-Blicharz et al. Table 2.
Order
Group
Species
Number sampled
Number positive
Percent positive
Anseriformes
Swans
Cygnus olor Cygus cygnus Anser albifrons Anser anser Anser fabalis undefined Anas platyrhynchos Anas querquedula Anas crecca Branta canadensis undefined Gallinago gallinago Larus ridibundus Larus canus Larus argentatus Sterna hirundo Sturnus vulgaris Columba livia Grus grus Fulica atra Phasianus colchicus Coturnix coturnix Perdix perdix Lyrurus tetrix Ciconia ciconia Falco tinnunculus Accipiter gentilis Aquila chrysaetos Dromaius novaehollandiae Ardea cinerea 28 defined
161 7 27 33 30 50 292 20 30 2 53 14 43 57 6 7 1 6 4 10 10 4 2 1 2 3 2 1 2 4 884
2 0 0 1 0 5 12 1 0 0 4 0 3 0 0 0
1.2 –a – 3.0 – 10.0 4.1 5.0 – – 7.5 – 7.0 – – – – – – – 30.0 – – – – – – – – – 3.5
Geese
Ducks
Charadriiformes
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Overview of the prevalence of avian coronaviruses in the samples studied.
Waders Gulls
Tern Passeriformes Columbiformes Gruiformes
Pigeon
Galliformes
Ciconiiformes Falconiformes Acciptriformes Casuariiformes Pelecaniformes Total: 11 a
0 0 0 3 0 0 0 0 0 0 0 0 0 31
No positive samples.
with each other, ranging from 90.0 to 99.6%, and were different from IBV and TCoV, with identities ranging from 85.2 to 88.3%. They formed a separate cluster with the bootstrap value of 95% (Figure 2). Three strains of ACoVs (ACoV/Duck/PL-MW283/2009, ACoV/Duck/PL-MW284/ 2009 and ACoV/Goose/PL-MW162/2009) were 98.1 to 99.6% similar to each other and they originated from the birds caught at different times but at the same ornithological
observation site (Dolnoslaskie). On the other hand, the gene 3 sequence of ACoV/Mallard/PL-MW150/2009 diverged from the other, with a nucleotide identity of 90.0 to 90.7%; this originated from the birds caught about 500 km away from Dolnoslaskie. With regard to the N gene (about 840 nucleotide), eight of the analysed fragment CoV strains fell into two different groups in the phylogenetic tree and the nucleotide
Table 3. Details of coronaviruses from wild bird samples that were molecularly characterized in this study.
Strain of avian CoV (species/unique number/year) 1 2 3 4 5 6 7 8 9 10 a
Mallard/PL-MW150/2009 Goose/PL-MW162/2009 Duck/PL-MW283/2009 Duck/PL-MW284/2009 Mallard/PL-MW345/2009 Duck/PL-MW371/2009 Bean goose/PLMW434/2009 Bean goose/PLMW435/2009 Mallard/PL-MW123/2010 Mallard/PL-MW272/2011
N gene
3′ UTR
+ + + + – – –
– + + + – + +
– + + + + + +
+
–
+
+
– –
– –
+ +
– +
Place/part of Poland
Sample date
Zelwa lake/north eastern Bukowka reservoir/south western Bukowka reservoir/south western Bukowka reservoir/south western Stary Lubliniec reservoir/south eastern Bukowka reservoir/south western Bukowka reservoir/south western
29 April 6 May 2 October 2 October 23 October 4 November 16 December
–a – + + + – +
Bukowka reservoir/south western
16 December
Jeziorsko reservoir/central Zywieckie lake/southern
26 April 26 October
Negative result in respective RT-PCR protocol.
RdRp gene 3 gene
IBV-like viruses in wild birds
409
IBV/Conn46 1983 (FJ904718) TCoV/TX-GL/01 (GQ427174) IBV/Cal557/2003 (FJ904715) IBV/Ark DPI (GQ504720) IBV/Georgia 1998 8p(GQ504722) ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 IBV/Mass41 Vaccine (GQ504725) ACoV/Peafowl/GD/KQ6/2003 (AY641576) IBV/CH/LDL/101212 (JF828981) IBV/H120 (GU393335) TCoV/Inn-517/94 (GQ427175) TCoV ATCC (EU022526) IBV/ITA/90254/2005 (FN430414) TCoV/FR080147C (FN811145) TCoV/FR070341J (FN811144) TCoV/FR080183J (FN811146)
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ACoV/Mallard/PL-MW345/2009 ACoV/Quail/Italy/Elvia/2005 (EF446156) IBV/vaccine strain 4/91 (KF377577) ACoV/Duck/CH/HN/ZZ2004 (JF705860) IBV/SAIBK (DQ288927) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Pintail/PBA-10 (GU396668) ACoV/Pintail/PBA-124 (GU396673 ) ACoV/Glaucous-winged gull/CIR-66002 (GU396682) ACoV/Rock sandpiper/CIR-65824 (GU396689) ACoV/Rock sandpiper/CIR-65828 (GU396689) ACoV/Brent goose/KR69 (GU396677) ACoV/Brent goose/KR70 (GU396676) Beluga whale CoV/SW1 (EU111742) ACoV/Anas americana/100112 (JN788849) ACoV/Ardea cinerea/091223 (JN788874) ACoV/Anas crecca/091230 (JN788837) ACoV/Platalea minor/091127 (JN788788) ACoV/Trush/HKU12-600 (FJ376621) ACoV/Common-moorhen/HKU21-8295 (JQ065049) ACoV/White-eye/HKU16-6847 (JQ065044) Asian leopard-cat CoV (EF584908) ACoV/Magpie-robin/HKU18-chu3 (JQ065046) ACoV/Munia/HKU13-3514 (FJ376622)
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Figure 1. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the RdRp gene of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura twoparameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
homology between them was 89.9 to 100% (Figure 3). Seven of these were in the “793B-like” cluster, with a significance of 63% for the bootstrap value. Similarly to the RdRp gene, the N genes of Anatinae and Anserinae strains isolated at the same sampling site and at the same time were the most closely related and formed two distinct clusters with nucleotide homology between them of 95.2%. The strains ACoV/Goose/PL-MW162/2009 and ACoV/Duck/ PL-MW371/2009 were most closely related to the 793Blike 4/91 IBV strain (95.5 to 95.8%), the component of the second most frequently used vaccine in Poland. The eighth strain, ACoV/Mallard/PL-MW123/2010, was in the “QXlike” cluster together with strains from Italy and Sweden
despite their nucleotide homology being between 91.5 and 93.7%. The 3′UTR fragment sequences (approximately 266 nucleotides) of eight CoVs detected in wild birds in Poland clustered into two groups and the nucleotide homology between them was 98.8 to 100% (Figure 4). Three ACoVs (ACoV/Bean goose/PL-MW435/2009, ACoV/Bean goose/ PL-MW434/2009 and ACoV/Mallard/PL-MW345/2009) shared 100% nucleotide sequence identity to each other and with the sequence of the North American IBV/DE072 strain and the Chinese IBV/ck/CH/LJC/111,054 strain. Five additional CoV strains that gave positive 3′UTR amplicons had nucleotide homology between 99.6 and 100% and
410
K. Domanska-Blicharz et al. IBV/ITA/90254/2005 (FN430414) IBV/SWE/0658946/10 (JQ088078) ACoV/Partridge/GD/S14/2003 (AY646283) IBV/CK/CH/LLN/98I (EF602451) ACoV/Duck/DK/CH/HN/2004 (HM371091) IBV/SAIBK (DQ288927) ACoV/Mallard/PL-MW150/2009 ACoV/Goose/PL-MW162/2009 ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 IBV/V2-71 (DQ490216) IBV/vaccine strain 4/91 (KF377577) IBV/Georgia 1998 pass8 (GQ504722) IBV/Delaware 072 (GU393332) IBV/Arkansas Vaccine (GQ504721) IBV/Conn46 1996 (FJ904716) IBV/CK/CH/LTJ/95I (EF602448)
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TCoV/TX-GL/01 (GQ427174) IBV/Massachusetts (GQ504724) ACoV/Peafowl/GD/KQ6/2003 (AY641576) IBV/Beaudette CK (AJ311317) IBV/CH/LDL/101212 (JF828981) IBV/H120 (GU393335) Beluga whale CoV/SW1 (EU111742) ACoV/Common-moorhen/HKU21-8295 (JQ065049) ACoV/White-eye/HKU16-6847 (JQ065044) ACoV/Thrush/HKU12-600 (FJ376621) ACoV/Munia/HKU13-3514 (FJ376622) ACoV/Magpie-robin/HKU18-chu3 (JQ065046) ACoV/Sparrow/HKU17-6124 (JQ065045)
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Figure 2. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of gene 3 of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura two-parameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
grouped together with the 4/91 vaccine strain. Divergence at the nucleotide level between these two groups was 1.5%.
Discussion In the present study, the vast majority of birds tested belonged to Anseriformes (79.7%), and Anas platyrhynchos and Cygnus olor constituted 33% and 18.2% of all birds tested, respectively. The next largest group of bird orders represented were Charadriiformes and Galliformes, which made up 14.4% and 2% of birds tested, respectively. During the 4-year period, the presence of coronaviral RNA was detected in 3.5% of all wild birds examined, and in samples originating from the most represented bird orders. The detection rate was 3.5% in Anseriformes and 2.3% in Charadriiformes, but as high as 17.6% in Galliformes even though this bird order was the lowest represented. In previous studies, the average prevalence in wild birds was 1.6% in northern England, over 6% in the Bering Strait Area (Beringia) and 12.5% in Hong Kong and Cambodia (Hughes et al., 2009; Muradrasoli et al., 2010; Chu et al., 2011). However, these results could not reflect the real prevalence of CoVs due to the fact that all studies have used different molecular methods for CoV detection and also due
to the huge diversity of CoVs, meaning that some of them could be missed. Recently, the application of the pancoronavirus molecular method aimed at the RdRp sequence has allowed the identification of a new genus of avian CoV, Deltacoronavirus (Woo et al., 2009; Chu et al., 2011). These viruses were detected mainly in Ciconiiformes, Pelecaniformes, Charadriiformes and Passeriformes (superorder Neoaves) and also, but to a lesser extent, in Anseriformes (superorder Galloanserae). In contrast, the occurrence of gammacoronaviruses was reported predominantly in Galloanserae. The authors of this survey concluded that deltacoronaviruses could have a more stringent host specificity than gammacoronaviruses, among which frequent intergenus and interspecies transmission is observed (Chu et al., 2011). The method used in our study enabled the detection of gammacoronavirus of poultry (IBV and TCoV), so it focused on the detection of similar viruses in wild birds. Because of this, the demonstration of the highest prevalence of CoVs in wild birds from the Galliformes order is not surprising. Other CoVs were detected in wild waterfowl of the orders Anseriformes and Charadriiformes such as swans, graylag geese, mallards, garganeys and black-headed gulls, but the detection rate in these orders was lower. A recent Chinese survey of domestic fowl
IBV-like viruses in wild birds
411
ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 ACoV/Duck/PL-MW371/2009 ACoV/Goose/PL-MW162/2009 IBV/vaccine strain 4/91 (KF377577) TCoV/France/ FR080147c (FN665665) TCoV/France/FR080183j (FN665666) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Mallard/PL-MW272/2011 IBV/ITA90254/2005 (FN430414) ACoV/Mallard/PL-MW123/2010 IBV SWE/0658946/10 (JQ088078) IBV/CH/LDL/101212 (JF828981 ) IBV/H120 (GU393335) IBV/Cal557/2003 (FJ904715 ) IBV/Georgia 1998 (GQ504722)
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IBV H52 (EU817497) ACoV/Peafowl/GD/KQ6/2003 (AY641576) TCoV ATCC (EU022526) IBV/ArkDPI (FJ904719) IBV/Conn46/1983 ((FJ904718) TCoV/In-517/94 (GQ427175) TCoV/TX-GL-01 (GQ427174) IBV/SAIBK (DQ288927) Beluga whale CoV/SW1 (EU111742) ACoV/Bulbul/HKU11-934 (FJ376619) ACoV/Thrush/HKU12-600 (FJ376621) ACoV/Munia/HKU13-3514 (FJ376622) Asian leopard cat CoV/Guangxi/F230/2006 (EF584908)
0.1
Figure 3. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the N gene of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using the neighbour-joining method and the Kimura two-parameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
revealed the presence of the N gene of IBV-like viruses in 4.25% of chickens but also in 0.80% of ducks and 1.82% of geese. These CoVs grouped together with IBV from chickens in different clades of the phylogenetic tree, resulting from the analysis of N gene sequences (Chen et al., 2013). The observed differences in avian CoV prevalence in wild birds could also result from the geographical location of sampling and/or the years of research. Results of Norwegian studies revealed that the percentage of CoVpositive birds in 2004 was 38%, and in 2003 was only 16% (Jonassen et al., 2005). In our study, the yearly CoV prevalence was similar and fluctuated around 3.5%, with the lowest value of 2.5% in 2010 and the highest value of 4.9% in 2011. Additionally, similar to avian influenza virus, the frequency of CoV detection may also be connected with the season. We investigated the seasonal pattern of CoV circulation in wild birds during the whole year, and two waves of infection were found. The first wave, between 3.4 and 3.6%, occurred in late autumn (October to December) and this pattern is similar to avian influenza virus circulation in wild birds (Munster et al., 2007). All 14 CoVinfected birds in this season belonged to Anseriformes. This wave of CoV prevalence may be connected with autumn migration when young and immunologically naive birds could be carrying CoV or become infected with local
circulating CoV. The second wave of CoV prevalence, with a rate of 8 to 16.4%, was observed in late spring (April to May). Among 17 CoV-infected birds, six belonged to Charadriiformes and Galliformes and 11 to Anseriformes. This epidemiological cycle of CoV coincides with the breeding season and may be the result of the increased need for food, and also possible contact with domestic poultry. Most positive wild birds originated from the areas surrounding artificial water retention, such as the Bukówka reservoir situated in the valley of the river Bóbr. Many poultry farms are located in this geographic region. There are also a few villages where backyard poultry are held, thus creating favourable conditions for contact with wild birds. No information was collected regarding clinical signs or body parameters in the CoV-infected birds. We therefore reached no conclusion as to whether these infections had any influence on health status. Previous reports are ambiguous, as numerous CoV-positive wild birds were described as healthy (Jonassen et al., 2005; Liu et al., 2005; Sun et al., 2007; Hughes et al., 2009). However, CoV infection in wild birds reported in other papers had a clinical significance for the birds (Jonassen et al., 2005; Gough et al., 2006; Qian et al., 2006; Circella et al., 2007). Out of 31 positive results in the rRT-PCR specific for the 5′UTR genome fragment, only 10 gave positive results in
412
K. Domanska-Blicharz et al. ACoV/Anas/p42/2005/GBR (FJ490199) ACoV/Oystercatcher/p17/2006/GBR (FJ490198) ACoV/Red knot/p60/2006/GBR (FJ490197) TCoV-ATCC (EU022526) ACoV/Bean goose/PL-MW434/2009 ACoV/Bean goose/PL-MW435/2009 ACoV/Mallard/PL-MW345/2009 IBV/DE072 (AF203002) TCoV/TX-1038/98 (GQ427176) IBV/NGA/A116E7/2006 (FN430415) IBV/Beaudette US (AJ311362) IBV/ArkDPI11 (EU418976) IBV partridge/GD/S14/2003 (AY646283) ACoV/Partridge/GD/S14/2003 (AY646283) IBV/vaccine strain 4/91 (KF377577) IBV/ITA/90254/2005 (FN430414) IBV/H120 (GU393335)
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IBV TCoV/TX-1038/98 (GQ427176) IBV CK/SWE/0658946/10 (JQ088078) ACoV/Mallard/PL-MW272/2011 ACoV/Goose/PL-MW162/2009 ACoV/Anas/UK/p33/2005 (FJ490195) ACoV/Duck/PL-MW283/2009 ACoV/Duck/PL-MW284/2009 ACoV/Duck/PL-MW371/2009 ACoV/Whooper swan/UK/p3/2005 (FJ490193) IBV/strain Mass41 (GQ504725) ACoV/Peafowl/GD/KQ6/2003 (AY641576) Beluga whale CoV/SW1 (|EU111742) ACoV/Thrush CoV/HKU12-600 (FJ376621) ACoV/Common-moorhen CoV/HKU21-8295 (JQ065049) ACoV/Magpie-robin CoV/HKU18-chu3 (JQ065046) ACoV/White-eye CoV/HKU16-6847 (JQ065044)
0.1
Figure 4. Consensus phylogenetic tree resulting from the analysis of the nucleotide sequences of the 3′UTR fragment of CoV detected in wild birds sampled in the territory of Poland (black dots) and other gammacoronaviruses and deltacoronaviruses (accession numbers in parentheses). Deltacoronaviruses highlighted in italics. The trees were computed using neighbour-joining method and the Kimura twoparameter model. The significance of the tree topology was assessed by 1000 bootstrapping calculation.
RT-PCRs aimed at other fragments of the IBV-like genome. To our knowledge, this is the first report of the analysis of different fragments of the IBV-like genome detected in wild bird populations. Most of the studies on the prevalence and molecular characterization of CoV in wild birds were based on the detection of just one genome fragment. Usually this was the RdRp gene, but others such as the N gene or the 3′UTR fragment have also been investigated (Jonassen et al., 2005; Hughes et al., 2009; Muradrasoli et al., 2010). In our study, we focused on four IBV genome fragments—RdRp, the 3 gene, the N gene and the 3′UTR fragment—with most positive amplicons being obtained in PCRs with primers directed to the N gene and 3′UTR fragment, and the least positives in the case of PCRs aimed at genes 3 and RdRp. The reason for failures in other RTPCR attempts can include either a very high sensitivity of the rRT-PCR method for CoV detection or, more probably, the gene sequence variability of the CoV detected, so that the primers used did not match them. The four phylogenetic trees shown here, based on the RdRp, genes 3 and N, and the 3′UTR, show that viruses from wild birds segregated with other avian CoVs,
including IBV, TCoV and viruses from quail, peafowl and duck, indicating that these viruses are IBV-like gammacoronaviruses. However, for each analysed genome fragment, CoV strains from wild birds shared a different evolutionary branch that resulted in a different location on the phylogenetic trees. Most gene fragments detected seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX) (Domanska-Blicharz et al., 2006, 2007). CoVs detected in northern England originating from birds of the Anseriformes and Charadriiformes orders had genomic 3′UTR fragments phylogenetically related to IBV. Moreover, virus sequences from most CoV samples shared high homology with the IBV H120 vaccine strain. Only a few of these were divergent, and more clustered with the 3′UTR sequence of a North American IBV and a TCoV. CoVs similar to IBV H120 vaccine strain were also described in healthy, unvaccinated, domestic peafowl and wild peafowl in China, where their presence probably arose from widespread use of IBV vaccines in the local poultry population (Liu et al., 2005). The 3′UTR fragment of most of the CoVs analysed in this study also shared high
IBV-like viruses in wild birds
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nucleotide identity with the sequence of the IBV H120 vaccine strain, commonly used for the immunoprotection of commercial chickens in Poland. Interestingly, three of them had high nucleotide similarity to a previously identified and unusual CoV detected in northern England (Hughes et al., 2009). In conclusion, our results described the prevalence of CoVs in the wild bird population in Poland. The viruses detected are IBV-like gammacoronaviruses, which carry selected gene fragments of different lineages of IBV. The source of wild bird infections with IBV-like CoV was probably poultry, and IBV genome detection could suggest that wild birds might constitute the mobile genome deposit of the CoV carrying gene pool, even in a huge geographic territory. Also, the possibility of recombination between different CoV strains, when they infect the same wild bird host, could constitute the mixing vessel where new variants of CoV arise. Acknowledgements The authors would like to thank Lukasz Bocian from the Department of Epidemiology and Risk Assessment, National Veterinary Research Institute for his help with analysis of the data obtained.
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