This first fifth-generation scientific journal aims to spark a revolution in academic publishing
1
2
3
4
5
6
7
(Incomplete manuscript)
Surveillance and Taxonomic Analysis of the Coronavirus
Dominant in Pigeons in China
8
9
10
11
12
Authors
Qing-Ye Zhuang, Kai-Cheng Wang, Shuo Liu, Guang-Yu Hou, Su-Chun Wang, Wen-Ming Jiang, Jin-Ping Li,
Jian-Min Yu, Ji-Ming Chen
13
14
Author Affiliation(s)
China Animal Health and Epidemiology Center, Qingdao, 266032, China.
15
16
Corresponding Author(s)
Dr. Ji-Ming Chen (E-mail: chenjiming@cahec.cn; jmchen678@qq.com).
17
18
19
20
21
22
Subject Areas
Veterinary medicine; Epidemiology; Evolution; Genetics; Infectious diseases; Microbiology; Taxonomy; Virology;
Ecology.
Newpubli, 2015, 1, e0002 ● Page 1
23
Abstract
24
25
26
27
28
29
30
31
32
33
34
35
36
37
We reported recently that the coronaviruses dominant (i.e., mainly circulating) in ducks (DdCoVs) should represent a
novel species different from the one represented by the coronaviruses dominant in chickens (CdCoVs). In this study
we conducted a large-scale surveillance of coronaviruses in chickens, ducks, geese and pigeons using a conserved
RT-PCR assay. The surveillance demonstrated that CdCoVs, DdCoVs and the coronaviruses dominant in pigeons
(PdCoVs) belong to different lineages, and they are all prevalent in live poultry markets in some regions of China.
We further sequenced some genomic segments of three PdCoVs, and found that the genetic distances between
PdCoVs and DdCoVs or CdCoVs are large enough to separate PdCoVs into a novel species different from the ones
represented by DdCoVs or CdCoVs within the genus Gammacoronavirus, per the species demarcation criterion of
International Committee on Taxonomy of Viruses. Therefore, this study provided novel evidences to update the
taxonomy of the coronaviruses circulating in poultry, and shed novel insight into the genetic diversity, evolution and
distribution of the coronaviruses circulating in domestic birds.
Newpubli, 2015, 1, e0002 ● Page 2
Pigeon coronavirus
38
Results
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
Surveillance of coronaviruses (CoVs) in poultry
75
76
77
78
79
80
81
82
Analysis of the sequences of seven conserved domains
A total of 693 CoVs were identified from the 4411 swab samples detected through inoculation in embryonated
eggs followed by the conserved RT-PCR assay and the 604 feces or drinking-water samples detected directly using
the conserved RT-PCR assay.
As showed in Figure 1, phylogenetic analysis of the RT-PCR amplicons suggested that these 693 viruses could
be classified into four lineages corresponding to pigeon-dominant coronavirus (PdCoVs) (n = 157), -dominant
coronavirus (CdCoVs) (n = 430), duck-dominant coronavirus (DdCoVs) (n = 104), and goose-dominant coronavirus
(GdCoVs) (n = 2), respectively.
As showed in Figure 2, the positive percentage of PdCoVs in the samples of pigeons, 26.12% (146/559), was
significantly higher than that in the samples of ducks (0.55%, 8/1444), chickens (0.08%, 2/2518), geese (0.20%,
1/494), with P < 0.01 by the Chi-square test. These data suggested that PdCoVs mainly circulate in pigeons, and
circulate in ducks, chickens and geese at a relatively low prevalence.
The positive percentage of CdCoVs in the samples of chickens, 15.13% (381/2518), was significantly higher
than that in the samples of ducks (1.32%, 19/1444), geese (2.23%, 11/494), pigeons (3.40%, 19/559), with P < 0.01
by the Chi-square test. These data suggested that CdCoVs mainly circulate in chickens, and circulate in ducks, geese
and pigeons at a relatively low prevalence.
The positive percentage of DdCoVs in the samples of ducks (6.45%, 96/1444), was significantly higher than
that in the samples of chickens (0.00%, 0/2488), geese (0.40%, 2/494), pigeons (1.07%, 6/559), with P < 0.01 by the
Chi-square test, suggesting that DdCoVs mainly circulate in ducks, and circulate in chickens, geese and pigeons at a
relatively low prevalence.
The positive percentage of GdCoVs in the samples of geese, 0.40% (2/494), was significantly higher than that
in other avian samples (0.00%, 0/4521) in this study, with P < 0.01 by the Chi-square test. These data suggested that
GdCoVs mainly circulate in geese.
PdCoVs were found in 81.82% (9/11) of the provinces and 45.31 % (29/64) of the live poultry markets (LPMs)
where the samples were collected. CdCoVs were found in 90.91% (10/11) of the provinces and 62.5% (40/64) of the
LPMs where the samples were collected. DdCoVs were found in 90.91% (10/11) of the provinces and 40.63%
(26/64) of the LPMs where the samples were collected where the samples were collected. These data suggested that
PdCoVs, CdCoVs and DdCoVs are prevalent in LPMs in some regions in China.
Of the aforementioned 157 PdCoVs, 146 were from pigeon samples, and 11 were from non-pigeon samples. As
for the 146 pigeon-origin PdCoVs, 125 from pigeon feces samples, 15 were from pigeon swab samples, 6 from
pigeon drinking-water samples. The prevalence of PdCoVs in pigeon swab, feces and drinking-water samples were
5.12% (15/293), 50.61% (125/247) and 31.58% (6/19). All the collected pigeon feces and drinking-water samples
were found to be CoV negative when they were detected through inoculation in chicken embryonated eggs followed
by the conserved RT-PCR assay. This indicates that PdCoVs replicate poorly in chicken embryonated eggs. As for
the eleven non-pigeon-origin PdCoVs, two were from chicken swab samples, eight from duck feces samples, and
one from goose drinking-water samples.
As given in Table 1, the identity between three PdCoVs reported herein and twelve randomly selected CdCoVs
(ten from chickens, one from turkeys, and one from peafowl) and three DdCoVs in the combined amino acid (aa)
sequences of the seven conserved domains in the viral replicase gene, namely ADP-ribose-1-phosphatase (ADRP)
in nsp3, nsp5, nsp12, nsp13, nsp14, nsp15 and nsp16, is 83.4%–85.0% between PdCoVs and CdCoVs,
86.7%–89.8%between PdCoVs and DdCoVs, 85.2%–88.8% between CdCoVs and DdCoVs. In contrast, the
corresponding aa identity is 97.0%– 98.5% among the three PdCoVs, 94.5%–99.2% among the twelve CdCoVs, and
94.5%–99.2% among the three DdCoVs. Therefore, according to the sole species demarcation criterion of
Newpubli, 2015, 1, e0002 ● Page 3
Pigeon coronavirus
83
84
85
86
87
88
89
90
International Committee on Taxonomy of Viruses (ICTV) for CoVs that the viruses sharing more than 90% aa
sequence identity in these seven conserved domains belong to the same species, the PdCoVs, DdCoVs and CdCoVs
should belong to three different species.
Phylogenetic analysis of the sequences of the seven domains combined together, as showed in Figure 3,
suggested that the PdCoVs, DdCoVs and CdCoVs belong to different lineages. The PdCoVs, DdCoVs and CdCoVs
also belong to different lineages based on each of these seven conserved domains, with the exceptions that
DdCoV/DK/Guangdong/2014 is located in the same lineage with the 12 CdCoVs based on the nsp5 gene sequences
and the nsp16 gene sequences. The exceptions might be caused by genomic recombination.**
91
Discussion
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
This study showed that the genetic distances between PdCoVs, DdCoVs and CdCoVs are large enough to
separate them into different species in the genus Gammacoronavirus, according to the official criterion of ICTV. The
large-scale surveillance of this study revealed that PdCoVs, DdCoVs and CdCoVs mainly circulate in pigeons, ducks
and chickens, respectively, and they belong to different phylogenetic lineages. Therefore, classifying PdCoVs,
DdCoVs and CdCoVs into different species also meets the official definition of virus species of ICTV that a virus
species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological
niche.
Currently, Avian coronavirus is the designation of the species within Gammacoronavirus covering infectious
bronchitis viruses (IBVs) in chickens and related CoVs in turkeys and other birds. The designation has become
questionable because some CoVs in wild birds have been identified to be members of another genus
Deltacoronavirus. In this report, we further challenged the designation of Avian coronavirus as we provided solid
evidences in supporting that the CoVs dominant respectively in pigeons, ducks and chickens likely belong to
different species. These three potential species can be designated as Pigeon-dominant coronavirus,
Chicken-dominant coronavirus and Duck-dominant coronavirus, and the designations allow that these CoVs may
also circulate in turkeys, geese, peafowl, or other birds at a relatively low prevalence.
Our surveillance results indicate that PdCoVs likely replicate poorly in chicken embryonated eggs. Therefore,
the detection procedure with inoculation in chicken embryonated eggs is not suitable for the detection of PdCoVs,
although it is suitable for surveillance of avian influenza viruses, Newcastle viruses and CdCoVs. Consequently, the
prevalence of PdCoVs in pigeon swab samples detected with inoculation in chicken embryonated eggs could be
greatly underestimated in this study.
Our surveillance results suggest that PdCoVs, CdCoVs and DdCoVs are of high prevalence at LPMs in some
regions in China. Therefore, LPMs likely play an important role in the circulation of a diversity of CoVs in poultry,
as they do in the circulation of avian influenza viruses.
In conclusion, this study shed novel insight into the genetic diversity, distribution, evolution and taxonomy of the
CoVs in poultry.
Newpubli, 2015, 1, e0002 ● Page 4
Pigeon coronavirus
117
Tables
118
119
120
Table 1. The genetic distances between CdCoVs, DdCoVs and PdCoVs in seven
conserved genomic domains.
Genetic distance ( x  s)
CdCoVs
DdCoVs
PdCoVs
CdCoVs
DdCoVs
PdCoVs
96.0%  1.0%
86.5%  1.0%
84.1%  0.4%
95.9%  2.6%
88.6%  1.1%
97.5%  0.8%
121
122
123
Newpubli, 2015, 1, e0002 ● Page 5
Pigeon coronavirus
124
Figures
125
126
127
128
129
Figure 1. Phylogenetic relationship among the CoVs identified through a large-scale surveillance based on the sequences
in the replicase gene amplified by the conserved RT-PCR.
Newpubli, 2015, 1, e0002 ● Page 6
Pigeon coronavirus
130
131
132
133
134
135
136
137
Figure 2. The positive percentage of PdCoVs, CdCoVs, DdCoVs and GdCoVs in pigeon, chicken, duck and goose
samples.
Newpubli, 2015, 1, e0002 ● Page 7
Pigeon coronavirus
138
139
140
141
142
143
144
145
146
Figure 3. Phylogenetic relationships of 18 CoVs based on the sequences of seven conserved domains in their genomes.
The first tree was based on the sequences of the seven domains combined together, and the remaining trees were based on
the sequences of each of the seven domains.
Newpubli, 2015, 1, e0002 ● Page 8
Pigeon coronavirus
147
Statements
148
149
150
151
152
153
154
155
156
157
158
Ethics
159
160
161
162
Competing Interests
163
164
165
Data Sharing
166
167
168
169
Funding
170
171
172
173
174
175
176
177
Copyright
The authors declare herein that they have not conducted plagiarism, falsification or dual submission as for this
article, and that they have been aware of and complied with the ethical requirements of Newpubli on authorship,
human rights, animal welfare, biosecurity and dual use of research.
The authors also declare herein that this study was conducted in strict accordance with the recommendations in
the Guide for the Care and Use of Laboratory Animals of China Animal Health and Epidemiology Center. The feces
samples, drinking-water samples and swab samples from poultry farms, backyard flocks and live bird markets, were
all collected with permission given by multiple relevant parties, including the Ministry of Agriculture of China,
China Animal Health and Epidemiology Center, the relevant veterinary section in the provincial and county or city
government, and the owners of the relevant birds.
The authors declare herein that no competing interests exist in this article, except that some authors are editors
of Newpubli. Newpubli has established the mechanism using software to keep the rating of each peer reviewer on the
value of each article blind to anyone and unable to be changed by anyone.
The authors declare herein that all data underlying the findings or conclusions of this article and its preprint are
fully available without restriction.
This work was supported by the Avian Influenza Surveillance Program of the Ministry of Agriculture, and the
Sci-tech Basic Work Project of the Ministry of Science and Technology (SQ2012FY3260033) in China. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The copyright of this article and its preprint completely belongs to its authors who allow anyone to read,
download, save, copy and print this article or its preprint, and use the metadata of this article related to index, search
and citation, without any restriction; the authors declare that any part of this article and its preprint cannot be used
without appropriate citation.
Newpubli, 2015, 1, e0002 ● Page 9