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\begin{document}

\title{Identification and Genomic Characterization of Two Novel Porcine
Circovirus Like Virus Strains Associated with Severe Diarrhoea and
Hemorrhagic Enteritis in Piglets in China}



\author[1]{Xianhui Liu}%
\author[1]{Xinming Zhang}%
\author[2]{Hanqin Shen}%
\author[3]{Kaiqi Lian}%
\author[1]{Jihao Zheng}%
\author[1]{Ge Xu}%
\author[4]{Leyi Zhang}%
\author[4]{Yanling Liu}%
\author[4]{Linyang Yu}%
\author[1]{Pengfei Zhang}%
\author[4]{Jianguo Dong}%
\author[5]{Changxu Song}%
\affil[1]{South China Agriculture University}%
\affil[2]{Wen’s Foodstuff Group Co. Ltd}%
\affil[3]{Anyang Institute of Technology}%
\affil[4]{Affiliation not available}%
\affil[5]{South China Agriculture UNiversity}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
The diarrhoea of pigs, in particular, for the newborns, is very harmful
to the pig industry. Porcine circovirus-like virus (Po-Circo-like (PCL)
virus) is a circular replication-associated protein (Rep)-encoding
single-stranded (CRESS) DNA virus. Two PCL virus strains, with severe
diarrhoea and hemorrhagic enteritis, have been found in two different
pig farms in Guangdong province, China. Subsequently, the full genomes
of two strains (PCL virus GD06 and PCL virus GD09) were sequenced. The
two PCL viruses contains 3942 nucleotides and 3925 nucleotides, which
vary from the genomes of other PCL virus strains with 3912, 3923, and
3942 nucleotides. Besides, the nucleotide identities between two strains
and other strains of PCL viruses and Bo-Circo-like virus/CH were
78\%-89\%. A multiple sequence alignment of these strains showed a
similarity of 86.2\%-94.4\% for the Rep gene sequence and 89.4\%-97.7\%
for the Rep protein sequence. This study found that 9.5\% (4/42) of
diarrhoea samples and 11.8\% (2/17) of pig farms were positive for PCL
virus, suggesting that PCL virus may already be widespread in Pig farms
in China. Further research on the pathogenicity and epidemiology of PCL
virus is required.%
\end{abstract}%



\sloppy


\textbf{Identification and Genomic Characterization of Two Novel Porcine
Circovirus Like Virus Strains Associated with Severe Diarrhoea and
Hemorrhagic Enteritis in Piglets in China}

Xianhui Liu\textsuperscript{1}, Xinming Zhang\textsuperscript{1}, Hanqin
Shen\textsuperscript{2}, Kaiqi Lian\textsuperscript{3}, Jihao
Zheng\textsuperscript{1}, Ge Xu\textsuperscript{1}, Leyi
Zhang\textsuperscript{1}, Yanling Liu\textsuperscript{1}, Linyang
Yu\textsuperscript{1}, Pengfei Zhang\textsuperscript{1}, Jianguo
Dong\textsuperscript{4}, Changxu Song\textsuperscript{1, *}.

Corresponding author: Changxu Song.

Email addresses: cxsong2004@163.com, cxsong@scau.edu.cn.

\textsuperscript{1}College of Animal Science \& National Engineering
Center for Swine Breeding Industry, South China Agriculture University,
Guangzhou, 510642, China

\textsuperscript{2}Wen's Foodstuff Group Co. Ltd, Guangdong Enterprise
Key Laboratory for Animal Health and Environmental Control, Yunfu,
527439, China

\textsuperscript{3}School of Biotechnology and Food Science, Anyang
Institute of Technology, Anyang, 455000, China

\textsuperscript{4}School of~Animal Husbandry and~Medical Engineering,
Xinyang Agriculture and~Forestry University, No. 1 North Road, Pingqiao
District, Xinyang~464000, China

\textbf{Abstract}

The diarrhoea of pigs, in particular, for the newborns, is very harmful
to the pig industry. Porcine circovirus-like virus (Po-Circo-like (PCL)
virus) is a circular replication-associated protein (Rep)-encoding
single-stranded (CRESS) DNA virus. Two PCL virus strains, with severe
diarrhoea and hemorrhagic enteritis, have been found in two different
pig farms in Guangdong province, China. Subsequently, the full genomes
of two strains (PCL virus GD06 and PCL virus GD09) were sequenced. The
two PCL viruses contains 3942 nucleotides and 3925 nucleotides, which
vary from the genomes of other PCL virus strains with 3912, 3923, and
3942 nucleotides.~Besides, the nucleotide identities between two strains
and other strains of PCL viruses and Bo-Circo-like virus/CH were
78\%-89\%. A multiple sequence alignment of these strains showed a
similarity of 86.2\%-94.4\% for the Rep gene sequence and 89.4\%-97.7\%
for the Rep protein sequence. This study found that 9.5\% (4/42) of
diarrhoea samples and 11.8\% (2/17) of pig farms were positive for PCL
virus, suggesting that PCL virus may already be widespread in Pig farms
in China. Further research on the pathogenicity and epidemiology of PCL
virus is required.

Keywords: Po-Circo-like virus, diarrhoea, sequence characterization,
widespread

Declarations

Funding

This study was supported by the National Key Technologies R\&D Program
(2015BAD12B02-5), Guangzhou City Project (201508020062).

Conflicts of interest/Competing interests

The authors declare no competing interests.

Availability of data and material

The authors show that it is credible

Code availability

The authors show that it is available

Ethics approval

This work is approved.

Consent to participate

The authors approve that the study is suitable for participation

Consent for publication

The authors agree that the study is publishable

Authors' contributions

Conceived and designed the experiments: CX S. Performed the
experiments:XH L, XM Z, KQ L, HQ S, LY Z, JH Z and G X. Analyzed the
data: XH L, YL L, LY Y, PY Z and JG D. Contributed
reagents/materials/analysis tools: CX S. Worte the paper: XH L and CX S.

1 INTRODUCTION

In 2011, a novel pig-derived circular replication-associated protein
(Rep)-encoding single-stranded (CRESS) DNA virus was discovered in pig
faces and named PCL virus (porcine circovirus-like virus) {[}1{]}. The
CRESS DNA viruses contain different genomic structures and are widely
present in diverse ecosystems. CRESS DNA virus has seven family members:
Circovidae, Nanoviridae, Smacoviridae, Genomoviridae, Bacilladnaviridae,
Kirkoviridae and Geminiviridae. The family Ciroviradae includes two
genera: Cyclovirus and Circovirus, causing a wide range of clinical
symptoms in humans, mammals, and birds{[}2{]}. A novel CRESS DNA virus
named Bo-Circo-like virus from a calf with severe haemorrhagic enteritis
has recently been detected in China{[}3{]}, and three porcine
circovirus-like viruses in pigs with diarrhoea have been found in
Guangxi, China{[}4{]}.

Porcine diarrhoea is one of the primary causes of piglet death, which
results in severe nutrition absorption and slow growth in pigs, leading
to substantial economic losses. Newborn piglets are highly vulnerable to
some enterovirus infections, causing severe diarrhea, enteritis and
vomiting. Enteric pig viruses include porcine epidemic diarrhoea virus
(PEDV), porcine delta coronavirus (PDCoV), porcine transmissible
gastroenteritis virus (TGEV), swine acute diarrhoea syndrome coronavirus
(SADS-CoV), porcine sapelovirus (PSV), porcine rotavirus (RV), porcine
picobirnavirus (PBV), porcine circovirus 4 (PCV4), porcine teschovirus
(PTV), porcine kobuvirus(PKV), porcine bocavirus(PBoV), porcine
sapovirus (SaV), porcine circovirus-like virus(PCL virus) and porcine
norovirus (NoV), was detected at porcine farms in China{[}4-13{]}.

In June, 2020, a fattening pig farm in Maoming, Guangdong province,
suffered from diarrhoea and wasting. Subsequently, PCL virus and PBoV
were found in clinical samples. In September, 2020, piglets suffered
from severe diarrhoea, enteritis and vomiting at a large-scale pig farm
in Qingyuan, Guangdong province. Then, we identified microbial pathogens
associated with diarrhoea, and only PCL virus was detected in the
diarrhoea samples.

These findings have helped us to understand the status of intestinal
infection in the Chinese pig population and also prompted us to
accelerate research into pathogenesis and epidemiology of the PCL virus.

2 MATERIALS AND METHODS

2.1 Clinical samples collection

In this study, 42 Clinical samples with severe hemorrhagic enteritis,
diarrhoea, lymphadenopathy, loss of appetite and vomiting of piglets
including intestinal tissue or faces, were obtained from 17 swine farms
in different regions of Guangdong province, China from January to
September 2020 and stored at -80.

2.2 DNA/RNA extraction of viruses

Clinical samples obtained were ground with double resistance PBS and
repeatedly freeze-thawed three times. The viral DNA/RNA is then
extracted using a viral nucleic acid extraction kit.

2.3 PCR detection

The primers of Viruses including PEDV, PDCoV, TGEV, RV, SADS-CoV, PTV,
PKV, PBV, PBoV, SaV, PSV, NOV and PCV4~have been found from the
literature{[}4-13{]}. 5 prime pairs were designed based on the reference
sequence of the PCL virus 21 and 22 strain determined in
the~United~States and the PCL virus GX14, GX15 and GX19 detected in
China (Table 1). PCR products were first isolated and identified by
agarose gel electrophoresis, and then obtained using a gel recovery kit
and cloned into a blunt-T plasmid. Whereas the ligands were transformed
into DH-5\selectlanguage{greek}α \selectlanguage{english}cells for gene cloning. The bacterial liquid was identified
and successfully connected to the bacterial liquid for sequencing.

2.4 DNA and amino acid sequences analysis

The complete gene sequences of PCL viruses obtained in this study have
been uploaded to Genbank with the accession numbers MW166350 and
MW166351. The genome lines were assembled using Lasergene. Subsequently,
all arrangements were further aligned with MegAlign (Lasergene) using
the ClustalW alignment method. A phylogenetic tree was built using the
maximum likelihood method with 1000 bootstrap replicates in MEGA7
software.

3 RESULTS AND DISCUSSION

CRESS viruses can infect a wide range of animals, even plants, and
mosquitoes. These viruses are found in pigs include PCV1, PCV2, PCV3,
PCV4, porcine circovirus-like virus P1 and PCL virus{[}11,14-16{]}.
PCV2,PCV3, porcine circovirus-like virus P1 and PCV4 have been
associated with clinical diseases in pig farms known as PCV-associated
disease (PCVAD) {[}11,14-16{]}. At present, PCV2 and PCV3 are widely
popular in the global pig industry and have caused substantial economic
losses{[}14,17{]}. PCL virus is very similar to PCV, and both have a
circular genome, however, PCL virus does not have a typical capsid
protein (Cap). The epidemiology of PCL virus has rarely been reported in
China were rarely reported.

In this study, two complete PCL virus genes were identified, and their
gene characteristics and genetic evolution were further analyzed. Two
clinical samples out of all 42 samples (9.5\%) and two pig farms out of
all 17 farms (11.8\%) were positive for the PCL virus. The PCL virus
GD06 were determined from fattening pigs with severe diarrhoea and loss
of appetite in Maoming, China. The PCL virus GD09 were detected from
newborn piglets (1 week to 4 weeks) associated with severe diarrhoea,
hemorrhagic enteritis and vomiting in Qingyuan, China. Some important
enterovirus (PEDV, PDCoV, TGEV, RV, SADS-CoV, PTV, PKV, PBV, PBoV, SaV,
PSV, NOV and PCV4) have been re-tested on PCL positive samples as shown
in table 2.

Further studies shown that PCL positive sample GD06 was co-infected with
PBOV. Enteritis and diarrhoea primarily occurred in piglets, but severe
diarrhoea, loss of appetite, and significant reduction in average daily
weight gain occurred in fat pigs, speculating the cause of co-infection
of these viruses. Besides, only PCL virus was found in sample GD09, PCL
virus infected Piglets in the delivery room were associated with severe
diarrhea and enteritis, whereas onset age of piglets ranged from 1 week
to 4 weeks. Anatomy of diseased piglets found small intestine mucosa
abscission, and intestinal mucosal lymph node enlargement. There was a
higher incidence of morbidity, while lower mortality among piglets as a
result of relief of symptoms by breeder's saline rehydration used in
piglets with diarrhea. It is well known that damage to intestinal mucosa
caused by diarrhoea or enteritis is harmful to the nutrient absorption
and growth rate of pigs. Thus, further studies on the pathogenicity and
gene evolution of the PCL virus should be carried out.

Subsequently, two full-length genome sequences of PCL virus were
obtained using an overlapping PCR method. The PCL virus GD06 genome
sequence (3942 bp) containing 3942 nucleotides in length and the PCL
virus GD09(3925bp) with 3925 nucleotides in length, which differ with
the PCL virus GX14(3944 bp), GX15(3944 bp) and GX19(3944bp) detected in
China and the PCL virus 21(3921bp) and 22(3922bp) detected in the US.

PCVs have a stem-loop structure {[}18{]}, whereas PCL virus contains a
14-nucleotide stem loop{[}4{]}, which is essential for replicating
viruses. PCL virus GD06 contains the
sequence\textsuperscript{1322}GGGCAATTCTGCCC\textsuperscript{1335},
which is the same as GX14 and GX15. PCL virus GD09, however, had the
sequence\textsuperscript{1322}GGGCAAGTCTGCCC\textsuperscript{1335},
which includes a substitution in the loop of the PCL virus(T1328G) and
is the same as GX19. Moreover, the substitution effects in the loop of
the PCL virus(T1328G) on pathogenicity, replication, and infectivity
require further study. (Fig 1)\begin{CJK}{UTF8}{gbsn}、

\end{CJK}\selectlanguage{english}The genome-wide pairwise identities between the PCL virus GD06 and the
strains 21, 22, GX14, GX15 or GX19 of the PCL virus were 79\%, 78\%,
79\%, 80\% and 80\%, respectively. And surprisingly, the complete genome
of PCL virus GD06 sequences is similar to that of the Bo-Cir-like virus,
which was associated with severe hemorrhagic~enteritis, with 79\%
similarity. The full-genetic similarity between PCL virus GD09 and the
strains 21, 22, GX19, GX15 and GX14, of the PCL virus were 89\%, 88\%,
89\%, 89\%, and 89\%, respectively. Consistent with this, the PCL virus
GD202009 and Bo-Cir-like virus had a high full genome-wide similarity of
89\%. Besides, a multiple sequence alignment of these strains, including
GD06, GD09, 21, 22, GX19, GX15 and GX14 of the PCL virus and
Bo-Circo-like virus CH, showed a sequence similarity of 86.2\%-94.4\%
for the rep gene lines and sequence similarity of 89.4\%-97.7\% for the
Rep protein arrangements.

Phylogenetic trees have been constructed using 1000 bootstrap replicates
of maximum like-lihood method based on amino acid sequences of the Rep
protein and the Rep gene sequences to understand better the relationship
between these different strains involved in this study (Fig 2). The
phylogenetic trees have shown human fecal virus, Rodent circovirus,
Kirkovirus Equ1, Bo-Circo-like virus CH, and PCL viruses belong to
family Kirkoviriade, a dependent branch. PCV2 and PCV3, which have
caused great economic loss and difficulties to the pig industry, can be
found in other hosts (mammals and even mosquitoes), whereas circovirus
is widely distributed in hosts{[}14,15{]}. Hence, further
epidemiological research is required.

The strains of PCL virus GD06 and PCL virus GD09 had a close
relationship to strains of Bo-Circo-like virus CH and the strains of
GX19, GX15, GX14, 21 and 22 of the PCL viruses. Besides, a phylogenetic
tree based on the rep gene sequences showed that all viruses are divided
into two main genotypes (PCLa and PCLb){[}4{]}, two PCL virus strains in
this study have been clustered into the PCLa branch. (Fig 3). The study
suggested that the PCL virus epidemic strains in China might occur to
due to the PCLa branch, and there is no apparent variation in the
epidemic strain. More clinical disease samples will be obtained to
analyze the domestic and global prevalence of PCL in the future.

In this study, two strains of PCL virus associated with severe diarrhea
and hemorrhagic enteritis were found in two large-scale pig farms in
Guangdong province, China. The results showed that the strains of PCL
and Bo-Cir-like viruses found in China had a close similarity, and both
were found in the same branch. In order to ascertain the spread of the
PCL virus across species, further studies of the pathogenicity and
epidemiology of the PCL virus need to be carried out at the earliest.

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