Veterinary Microbiology 240 (2020) 108522

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

First detection and genomic characterization of porcine circovirus 3 in T

mosquitoes from pig farms in China
Zhuo Haa,b, Jin-Feng Lic, Chang-Zhan Xiea,d, Cheng-Hui Lie, Hong-Ning Zhouf, Ying Zhangc,
Peng-Fei Haoa,c, Fu-Long Nana,c, Jin-Yong Zhanga,d, Ji-Cheng Hana,e, He Zhanga, Xin-Yu Zhuanga,
Ying-Cheng Guog, Hui-Jun Lua,h,*, Ning-Yi Jina,b,h,*
a
Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun, 130122, China
b
College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
c
College of Veterinary Medicine, Jilin University, Changchun, 130012, China
d
College of Animal Science and Technology, Jilin Agricultural University, Changchun, 130118, China
e
Medical College, Yanbian University, Yanji, 133002, China
f
Yunnan Institute of Parasitic Diseases, 665000, China
g
Jilin Fengman Area Animal Prevention and Control Center, Jilin, 132013, China
h
Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou, 225009, China

A R T I C LE I N FO A B S T R A C T

Keywords: The porcine circovirus type 3 (PCV3) becomes an important causative agent of swine disease since its discovery
PCV3 in 2016. PCV3 infection exhibits a wide range of clinical syndromes causing substantial economic losses in swine
Mosquitoes industry. Previous studies have reported the detection of numerous known viruses including circovirus in
Pig farms mosquitoes. However, the transmission of PCV3 in field-caught mosquitoes remains largely unknown. This study
Complete genome
aims to detect PCV3 infection in mosquitoes and analyze its genomic characteristics. Here, we performed a PCR
Transmission vectors
to detect the PCV3 in 269 mosquito samples collected from pig farms located in Heilongjiang, Jilin, and Yunnan
provinces. The proportion of PCV3-positive mosquitoes was 32.0 % (86/269), ranging from 21.4%–42.5% at
farm level, which may imply that mosquito serves as a route of transmission for PCV3. To determine the possible
origin of PCV3 in mosquitoes, 80 pig serum samples were collected from the pig farms where mosquito sampling
was also performed. The proportion of PCV3-positive farms ranged from 15.0%–30.0 % in which infection of
positive pigs positively correlated with mosquitoes carrying the virus. Additionally, we sequenced the entire
genome of 6 strains of PCV3 in mosquitoes and 2 strains of PCV3 in pigs. Sequence analysis indicated a 100 %
nucleotide similarity between mosquito and pig viral isolates that were all collected from similar farms.
Phylogenetic analysis showed that PCV3 could be divided into two clades, PCV3a and PCV3b, and the PCV3
strains isolated in mosquitoes were distributed on the two clades. Our results demonstrate that mosquitoes may
serve as a potential transmission vector in the life-cycle of PCV3, revealing possible transmission routes of PCV3.

1. Introduction
Porcine circovirus (PCV) is a small non-enveloped virus that belongs
to the genus Circovirus of the family Circoviridae with a genome of
circular single-stranded DNA (Rosario et al., 2017). In 2016, a novel
circovirus, termed PCV type 3 (PCV3), was identified in the USA by
metagenomic sequencing from pigs with clinical signs characterized by
porcine dermatitis and nephropathy syndrome (PDNS), reproductive
failure, as well as cardiac and multisystemic inflammation thereby
causing a huge economic losses in the swine industry (Palinski et al.,
2017; Phan et al., 2016). Subsequently, PCV3 has been detected in
other countries that included China, Thailand, South Korea, Brazil and
some European countries (Faccini et al., 2017; Fux et al., 2018; Hayashi
et al., 2018; Kedkovid et al., 2018; Kwon et al., 2017; Shen et al., 2018;
Stadejek et al., 2017; Tochetto et al., 2018; Ye et al., 2018; Yuzhakov
et al., 2018). To avoid the spread of the disease and its underlying
economic impact, it is essential to understand the prevalence and
transmission of PCV3.
Like any other circoviruses, PCV3 has a circular single-stranded
DNA genome (1999 to 2001 nucleotides in length) (Fux et al., 2018; Ha
et al., 2018; Wen et al., 2018), which contains two main inversely ar-
ranged open reading frames (ORFs), namely ORF1 and ORF2. The ORF1

⁎
Corresponding authors at: Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun, 130122, China.
E-mail addresses: huijun_lu@126.com (H.-J. Lu), ningyik@126.com (N.-Y. Jin).

https://doi.org/10.1016/j.vetmic.2019.108522
Received 18 September 2019; Received in revised form 21 November 2019; Accepted 21 November 2019
0378-1135/ © 2019 Elsevier B.V. All rights reserved.
Z. Ha, et al.
encodes a 296 amino acid (aa) replicase protein (Rep) while the ORF2
encodes a 241 aa capsid protein (Cap) (Palinski et al., 2017; Phan et al.,
2016). The Cap is the major structural protein of circoviruses in which
amino acid comparison revealed this structure of PCV3 was only shared
by less than 50 % identity at the aa level when compared to those of any
reported circoviruses (Phan et al., 2016). This characteristic implies
that PCV3 is distinct from the previously reported circoviruses. Cur-
rently, Jiang et al. reproduced a PDNS-like disease in piglets by PCV3
infection, which recorded 40 % mortality a few days after viral in-
oculation (Jiang et al., 2019a). The data indicate that PCV3 as an
emerging pathogen causes a serious effect on pig production and
therefore needs to be investigated further.
Previous studies have shown that PCV3 existed in a large number of
hosts, such as dog, cattle, chamois, roe deer, and mouse, indicating a
possible cross-species transmission (Franzo et al., 2019; Jiang et al.,
2019b; Wang et al., 2019; Zhang et al., 2018). It is well-known that
mosquitoes play a crucial role in the global virosphere by acting as a
vector of many viral diseases (Hemingway et al., 2006; Yang et al.,
2012a,b). For example, the Japanese encephalitis virus (JEV) and the
Getah virus can be transmitted to humans through bites of mosquitoes
that have previously taken a blood meal from the infected pigs (Garjito
et al., 2018; Li et al., 2017). However, it is unknown whether mosquito
could serve as a vector or involved in maintaining and transmitting
PCV3. Therefore, the present study aims to investigate the presence and
possible origin of PCV3 in mosquitoes. We also aim to molecularly
characterize the PCV3 stains from mosquitoes captured in selected
provinces of China.
2. Materials and methods
2.1. Sampling
A total of 269 mosquito samples were captured between May and
July 2018 from seven different pig farms in Heilongjiang (farm A,
n = 42; farm B, n = 44), Jilin (farm C, n = 40; farm D, n = 49) and
Yunnan province (farm E, n = 28; farm F, n = 31; farm G, n = 35) in
China. Identification of sampled mosquito was performed in which four
different species was identified as Aedes vexans (n = 65), Anopheles si-
nensis (n = 64), Culex tritaeniorhynchus (n = 59) and Culex pipiens pal-
lens (n = 81). To determine the possible origin of PCV3 in mosquitoes, a
total of 80 serum samples from pigs were collected from similar mos-
quito sampling sites, i.e., Heilongjiang (farm A, n = 20; farm B, n = 20)
and Jilin (farm C, n = 20; farm D, n = 20) provinces.
2.2. DNA extraction and PCV3 detection
DNA extraction of the viral genome from the mosquito and pig
serum samples was performed using a Viral DNA Kit (OMEGA Bio-Tek,
Georgia, USA) according to the manufacturer’s instructions, Next, PCV3
detection by PCR was conducted as previously described (Ha et al.,
2018). Briefly, the amplification reaction was performed in a 20 μL final
volume with the following components: 1 μL DNA; 0.5 μL of each 20 μM
primer; 1 μL (2.5 mmol/L) dNTPs; 4 μL PCR buffer; 0.5 μL DNA poly-
merase (TransGen Biotech, Beijing, China); and, an appropriate volume
Table 1
The positive rate of PCV3 in mosquitoes and pigs from the same pig farm.
Geographical origin Pig farms Number of mosquito samples PCV3positiverate in mosquitoes
Heilongjiang Farm A 42 21.4
Farm B 44 40.9
Jilin Farm C 40 42.5
Farm D 49 28.6
Yunnan Farm E 28 32.1
Farm F 31 35.5
Farm G 35 22.9
Veterinary Microbiology 240 (2020) 108522
of ddH2O up to 20 μL. The PCR profile condition was as follows: 95 °C
for 2 min; 35 cycles of 95 °C for 20 s, 56 °C for 20 s, and 72 °C for 20 s;
and, a final extension at 72 °C for 5 min. The amplified products were
analyzed on 1.5 % agarose gels and positive amplicons were purified
and sequenced (Comate Biosciences Co., Jilin, China).
2.3. PCV3 genome sequencing
To obtain a complete genome of PCV3, a PCR primer pair designed
for sequencing was used as previously reported (Ha et al., 2018). In
brief, the PCR protocol was carried out in a 50 μL final volume that
consists of 4 μL DNA; 1 μL of each 20 μM primer; 2 μL (2.5 mmol/L)
dNTPs; 10 μL PCR buffer; 1 μL DNA polymerase (TransGen Biotech,
Beijing, China) ; and ddH2O up to 50 μL. The PCR profile conditions
were as follows: 95 °C for 2 min; 40 cycles at 95 °C for 20 s, 50 °C for
20 s, and 72 °C for 40 s; and, a final extension at 72 °C for 5 min. Each
fragment was amplified and purified using a Gel Extraction Kit (Bioer
Technology, Hangzhou, China) and cloned into a pEASY-Blunt vector
(TransGen Biotech, Beijing, China), three positive clones were obtained
for sequencing (Comate Biosciences Co., Jilin, China).
2.4. Bioinformatics analyses
For the phylogenetic analysis, we analyzed a total of 6 complete
PCV3 genome sequences derived from this study. For comparison and
as a reference sequence, we used different PCV3 strains isolated from
different geographical locations in China and other countries with
available data retrieved from the NCBI nucleotide database. Multiple
sequence alignments were performed using Lasergene software with the
Clustal W program implemented in DNAStar software. A phylogenetic
analysis of the complete PCV3 genome was reconstructed using MEGA
6.06 software with the p-distance-based, maximum-likelihood (ML),
and neighbor-joining (NJ) method with 1000 bootstrap replicates.
3. Results
3.1. PCV3 detection in mosquitoes
To examine the potential involvement of mosquitoes in the pre-
valence and transmission of PCV3, a total of 269 mosquito samples
were collected from seven pig farms in Heilongjiang, Jilin and Yunnan
province. The proportion of PCV3-positive mosquitoes was 32.0 % (86/
269), with 29.2 % (19/65) positivity in Aedes vexans, 37.5 % (24/64) in
Anopheles sinensis, 27.1 % (16/59) in Culex tritaeniorhynchus, and 33.3
% (27/81) in Culex pipiens pallens. At the province level, the proportion
of PCV3-positive mosquitos was 31.4 % (27/86) in Heilongjiang, 34.8
% (31/89) in Jilin, and 29.8 % (28/94) in Yunnan. At the farm level,
the proportion of PCV3-positive mosquitos was 21.4 % (9/42) in Farm
A, 40.9 % (18/44) in Farm B, 42.5 % (17/40) in Farm C, 28.6 % (14/
49) in Farm D, 32.1 % (9/28) in Farm E, 35.5 % (11/31) in Farm F and
22.9 % (8/35) in Farm G (Table 1).
To determine the correlation of PCV3-positive rate between mos-
quitoes and pigs, the PCV3-positive rate in pigs and mosquitoes from
the same farms located in Heilongjiang and Jilin province. The
Number of pig serum samples PCV3 positive rate in pigs
% (9/42) 20 15.0 % (3/20)
%(18/44) 20 25.0 % (5/20)
% (17/40) 20 30.0 % (6/20)
% (14/49) 20 15.0 % (3/20)
% (9/28) – –
% (11/31) – –
% (8/35) – –
2
Z. Ha, et al.
Fig. 1. The positive rate of PCV3 in mosquitoes and pigs from pig farms. (a) The positive rate of PCV3 in mosquitoes and pigs from farm A to farm D. (b) The positive
rate of PCV3 in mosquitoes was positively correlated with the positive rate of PCV3 in pigs from the same farm.
proportion of PCV3-positive pigs was 15.0 % (3/20) in Farm A, 25.0 %
(5/20) in Farm B, 30.0 % (6/20) in Farm C, and 15.0 % (3/20) in Farm
D (Table 1). These results indicate a high positive rate of PCV3 both in
mosquitoes and pigs. Furthermore, we found that the presence of PCV3
in mosquitoes was positively correlated with PCV3-positive detection in
pigs in the same pig farm, implying that mosquitoes might exacerbate
the spread of PCV3 (Fig. 1). Previous studies showed the extensive
PCV3 DNA was detected from different domestic or wild mammalian
hosts, as well as in ticks (Ouyang et al., 2019). Thus, we hypothesize
that mosquitoes may serve as an intermediate vectors that transmits
PCV3 within pig population or between other animals (Fig. 2).
3.2. Generation of whole-genome and sequence comparison of PCV3 in
mosquitoes
In order to study the gene characteristics of PCV3 derived from
mosquitoes, six complete sequences of the PCV3 genome were ampli-
fied and sequenced. We deposited in GenBank the complete genome
sequences of PCV3 isolated from mosquitoes under the accession
numbers MN431637-MN431642 (Table 2). The PCV3 genomes were
2000 nucleotides in length and the alignment of multiple sequences
within these six samples shared 98.3%–100% and 99.0%–99.7%
3
Veterinary Microbiology 240 (2020) 108522
Fig. 2. Mosquitoes may serve as potential
transmission vectors in the life-cycle of PCV3.
Previous studies have demonstrated that PCV3
was extensively circulated in domestic (pigs,
dogs and cattle) and wild (wild boar and deer)
animals. Mosquitoes may serve as transmission
vectors that circulate PCV3 between domestic
and wild host, even to humans.
nucleotide similarities at the ORF2 and complete genome sequences,
respectively. The samples also shared 97.8%–100% and 98.3%–99.7%
nucleotide similarities with the available PCV3 reference strains from
the NCBI GenBank at the ORF2 and complete genome sequences, re-
spectively (Table 3). These analyses thus revealed high genetic stability
of PCV3 strains.
Through multiple sequence alignment of capsid protein, we also
showed that the majority of variant PCV3 strains have five amino acid
mutations (A24 V, R24 K, S77 T, F104Y and I150 L) (Fig. 3). In addition,
some new mutations for the cap protein were observed in PCV3/CN/
JL/MO10/2018 (MN431640) strain (A108D and S156 G), PCV3/CN/
YN/MO4/2018 (MN431642), and PCV3/CN/HLJ/MO26/2018
(MN431637) strain (W186C) (Fig. 3). In previous reports, the majority
of variant PCV3 strains on the Cap protein from cattle and dogs were
observed in D124Y and V206A, respectively (Fig. 3) (Wang et al., 2019;
Zhang et al., 2018). However, the impact of the identified mutations on
capsid protein in PCV3 infections requires further investigation.
3.3. The origin of PCV3 in mosquitoes
Porcine serum samples were collected from similar pig farms to
trace the origin of PCV3 in mosquitoes. We detected and sequenced 2
Z. Ha, et al. Veterinary Microbiology 240 (2020) 108522

Table 2
Summary of obtained PCV3 strains in this study.
No. GenBank accession no. Strain name Geographic location Collection date Host

1 MN431637 PCV3/CN/HLJ/MO26/2018 Heilongjiang 2018 mosquito

2 MN431640 PCV3/CN/JL/MO10/2018 Jilin 2018 mosquito
3 MN431639 PCV3/CN/YN/MO1/2018 Yunnan 2018 mosquito
4 MN431638 PCV3/CN/YN/MO2/2018 Yunnan 2018 mosquito
5 MN431641 PCV3/CN/YN/MO3/2018 Yunnan 2018 mosquito
6 MN431642 PCV3/CN/YN/MO4/2018 Yunnan 2018 mosquito
7 MN431643 PCV3/CN/HLJ/p6/2018 Heilongjiang 2018 pig
8 MN431644 PCV3/CN/JL/p8/2018 Jilin 2018 pig

complete sequences of PCV3 strains in Heilongjiang (MN431643) and
Jilin (MN431644) province (Table 2). The results of whole-genome
sequencing alignment analysis revealed that PCV3 strains isolated in
pigs, sharing 100 % nucleotide identity with mosquitoes captured in the
same pig farms. Therefore, the results revealed that PCV3 strains in
mosquitoes derived from pigs.
3.4. Phylogenetic analysis of PCV3 in mosquitoes
To get a better understanding of the genetic relationship and evo-
lution of PCV3 in mosquitoes, the NJ and ML phylogenetic trees were
performed to reconstruct the phylogenies using the complete genome
sequences of PCV3. A total of 56 complete genome sequences available
in the NCBI database were compared with the 6 complete genome se-
quences (MN431637-MN431642) from mosquitoes where PCV3 was
divided into different clades. Our analysis indicated that PCV3 strains
were divided into two distinct clades, PCV3a and PCV3b. The PCV3
strains identified in mosquitoes belonged to the PCV3a and PCV3b
clades. According to the phylogenetic tree, the 6 strains of PCV3 have a
close evolutionary relationship with the USA and South China isolates
(Fig. 4). Our results also revealed that the PCV3 strains have no re-
lationship with the geographical location.
4. Discussion
PCV3 is an emerging porcine circovirus that has been proposed to be
associated with different clinical presentations in infected pigs, in-
cluding reproductive failure, CT, PNDS, and multi-systemic
inflammation (Chen et al., 2017; Kedkovid et al., 2018; Palinski et al.,
2017; Phan et al., 2016; Qi et al., 2019). Different epidemiological
survey showed that PCV3 was widely circulated in pigs from different
countries or regions. Recently, typical clinical signs of PDNS-like dis-
ease in piglets were reproduced by inoculating an infectious PCV3 DNA
clones (Jiang et al., 2019a) of which demonstrate a huge economic
impact in swine industry after showing a substantial mortality rate
among infected pigs. However, the transmission route of PCV3 is un-
clear, for instance, whether mosquitoes serve as an important vector in
spreading the virus remains to be proven. In the present study, we
observed a high PCV3 proportion of positively infected mosquitoes
collected from pig farms. Mosquitoes are capable of transmitting a wide
range of pathogens that cause human and animal illnesses (Lindahl
et al., 2012). It has been demonstrated that porcine reproductive and
respiratory syndrome virus (PRRSV) and PCV2 could be detected in the
mosquito, thus serving as a mechanical transmission vectors (Otake
et al., 2002; Yang et al., 2012a,b). Studies on JEV and Getah virus
suggested viral replication within mosquito cells indicating that the
insect serve as a biological transmission vector (Garjito et al., 2018; Li
et al., 2017). Our results suggest that mosquitoes may play a role as
reservoirs for the PCV3 virus, posing a threat to the swine industry.
Notably, we found a positive correlation between PCV3 proportions of
positive mosquitoes and pigs. Together with the identical sequence
results, these findings might suggest the potential role of mosquitoes in
sustaining the transmission of PCV3 in the pig population. However,
this study did not demonstrate under control conditions that mosqui-
toes carrying PCV3 could infect pigs. Hence, further studies are needed
to determine whether mosquitoes serve as biological transmission

Table 3
Summary of reference PCV3 strains in this study.
No. GenBank Accession no. Strain name Geographic location Collection date Host

1 MH107161 PCV3/Shandong-01 China 2016 bovine

2 MH107163 PCV3/Shandong-03 China 2016 bovine
3 MH445393 PCV3/Nanjing/BALB/C2 China 2018 mouse
4 MH445394 PCV3/Nanjing/BALB/C4 China 2018 mouse
5 MH579747 PCV3/Wild boar/2018 Spain 2018 Sus scrofa
6 MH579739 PCV3/Wild boar/ 2010 Spain 2010 Sus scrofa
7 KT869077 PCV3/29160 USA 2015 pig
8 MG014376 PCV3/ DE55.1 Germany 2015 pig
9 MG595741 PCV3/HU/Szerencs/2017 Hungary 2017 pig
10 KY996345 PCV3/KU-1609 South Korea 2016 pig
11 LC269727 PCV3/GM2124-2/2016 Japan 2017 pig
12 MH327784 PCV3/COL/2018 Colombia 2018 pig
13 MF805724 PCV3/4332-7/Denmark Denmark 2017 pig
14 MG310152 PCV3/Thailand/PB01/17 Thailand 2017 pig
15 MG679917 PCV3/RU/SM17 Russia 2017 pig
16 MG765473 PCV3/SWE84/2004 Sweden 2004 pig
17 MF079253 PCV3/BR/RS/6 Brazil 2016 pig
18 MF162298 PCV3/IT/CO2017 Italy 2017 pig
19 MK347414 PCV3/JL53 China 2017 pig
20 MK347415 PCV3/HLJ3 China 2016 pig
21 MF677839 PCV3/JX-2/CH/2017 China 2017 pig
22 MH107161 PCV3/Shandong-01/2016 China 2016 pig
23 MG650176 PCV3/YN3/1996 China 1996 pig

4
Z. Ha, et al. Veterinary Microbiology 240 (2020) 108522

Fig. 3. Multiple amino acid alignment of the Cap protein of PCV3 isolates strains. The majority amino acid mutations in the Cap protein were indicated by a blue
block. A comparison of the point mutations of the Cap protein in this study was indicated by a red block. A comparison of the point mutations of the Cap protein in
dogs and cattle were indicated by a yellow block. (For interpretation of the references to colour in this Table legend, the reader is referred to the web version of this
article).

vector of PCV3 in pig population.
In circovirus, the Cap protein is the sole structural protein and thus
the major target of the host immune response. In the present study, the
ORF2 sequences of 6 PCV3 strains from mosquitoes shared
97.8%–100% nucleotide similarities with available PCV3 ORF2 se-
quences from the NCBI GenBank. This implies that PCV3 has high ge-
netic stability, which is concordant with findings from previous studies
(Qi et al., 2019). On the other hand, the ORF2 sequences of PCV2
strains from the NCBI GenBank only shared 89.0%–100% nucleotide
similarities (Zhai et al., 2014), suggesting that PCV3 has high genetic
stability than PCV2 strains. In a vaccination study using PCV2, the
variation of amino acid in its capsid protein has shown to facilitate
escape mechanisms from a vaccine-induced immune response
(Karuppannan and Opriessnig, 2017). In the present study, we found
five amino acid substitutions in Cap protein during the evolution of
PCV3, an observation identical to that reported by Fux et al. and Fu
et al. (Fu et al., 2018; Fux et al., 2018). In the previous reports, PCV3
strains from different hosts could be distinguished using the Cap pro-
tein, such that the amino acid at position 206 mutated from lysine in
pigs to alanine in dogs (Sun et al., 2019) and the amino acid at position
124 from aspartic acid in pigs to tyrosine in cattle (Wang et al., 2019).
In the present study, we observed new amino acid variations on the
capsid protein of PCV3 in mosquitoes but whether these mutations were
related to the pathogenicity of PCV3 requires further investigation. So
far, there is no existing fixed criterion for the classification of PCV3. Li
et al. provided a comprehensive genotype identification for PCV3 (Li
and Wang, 2018). In our study, we referred to the methods used by Li
et al. to divide the strains of PVC3 into different clades. Hence, PCV3
could be stably divided into two clades (PCV3a and PCV3b) based on
the complete coding sequences whereby the PCV3 strains isolated in
our study were distributed on the two clades. Furthermore, we found no
relationship between isolates in terms of regional distribution corro-
borating with the previous studies that showed no regional origin dis-
tribution among PCV3 strains (Fux et al., 2018; Liu et al., 2019; Sukmak
et al., 2019).
Previous studies demonstrate that PCV3 is extensively prevalent in
domestic pigs and wild boars (Liu et al., 2019; Prinz et al., 2019). In-
creasing evidence has shown that PCV3 is capable of cross-species
transmission to non-porcine hosts, such as dogs, cattle, deer and ticks
(Franzo et al., 2019; Wang et al., 2019; Zhang et al., 2018). Our se-
quencing results of the complete PCV3 genome revealed that PCV3
existed in mosquitoes, suggesting a potential reservoir for PCV3 trans-
mission. However, whether PCV3 could be transmitted from pigs to
other animals by mosquitoes remains unknown and needs further study.

5
Z. Ha, et al.
Fig. 4. Phylogenetic analysis of the PCV3 based on complete coding sequences. The tree was constructed using MEGA version 6.06 with 1000 bootstraps replicates
and a p-distance model. PCV3 strains are denoted by its accession number at GenBank, country of origin, and isolation year. The 6 PCV3 strains collected from
mosquitoes are indicated with a red triangle. (a) maximum likelihood method. (b) neighbor-joining method. (For interpretation of the references to colour in this
Table legend, the reader is referred to the web version of this article).
Using a nonhuman primate model, Kruger et al. showed that PCV3
could replicate in Baboon by transplantation the heart of a PCV3-po-
sitive donor pig (Kruger et al., 2019). In addition, the bioinformatics
analysis performed by Li et al. demonstrated that PCV3 might pose a
potential risk to public health (Li and Wang, 2018). Our results may
imply that PCV3 in mosquitoes might pose a threat to humans, em-
phasizing the essentiality of controlling mosquitoes to prevent a po-
tential outbreak of PCV3 disease among pig populations.
In conclusion, to our knowledge, this is the first report that PCV3 is
widely circulated in mosquitoes. We demonstrate that PCV3 in mos-
quitoes originates from pigs. However, whether mosquito could be a
potential vector that transmits PCV3 is something to be addressed in the
next step of the study.
Ethics approval and consent to participate
Pigs clinical samples used in this study had obtained a written
consent from farm owners and carried out in strict accordance with the
Animal Ethics Procedures and Guidelines of the People's Republic of
China. All of the animal protocols in this study were approved by the
Ethics Committee of Military Veterinary medicine Institute.
ORCID Z. Ha https://orcid.org/0000-0002-0072-627X
CRediT authorship contribution statement
Zhuo Ha: Data curation, Writing - original draft. Jin-Feng Li:
6
Veterinary Microbiology 240 (2020) 108522
Software. Chang-Zhan Xie: Software. Cheng-Hui Li: Software. Hong-
Ning Zhou: Software. Ying Zhang: Formal analysis. Peng-Fei Hao:
Formal analysis. Fu-Long Nan: Methodology. Jin-Yong Zhang:
Methodology. Ji-Cheng Han: Formal analysis. He Zhang: Formal
analysis. Xin-Yu Zhuang: Formal analysis. Ying-Cheng Guo: Formal
analysis. Hui-Jun Lu: Data curation, Funding acquisition, Writing -
original draft. Ning-Yi Jin: Funding acquisition.
Declaration of Competing Interest
All authors have declared no conflict of interest.
Acknowledgments
This work was supported by the following Grants: National Key
Research and Development Program of China (grant number
2017YFD0500101 and 2016YFD0500401), Yunnan Academician
Workstation (grant number 2018IC151). The funders did not play any
role in the design, conclusions or interpretation of the study.
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