Emergence of porcine circovirus‐like viruses associated with porcine diarrheal disease in China

Abstract Background The circular replication‐associated protein (Rep)‐encoding single‐stranded (CRESS) DNA virus emergence in diverse host has been associated with severe disease. Porcine circovirus‐like virus (Po‐Circo‐like [PCL] virus) is a CRESS DNA virus, the prevalence and pathogenicity of which are rarely studied. Methods We obtained two blood samples, four faecal samples, and two intestinal samples from a pig farm suffered from diarrheal disease in the delivery room in September 2020 and attempted to isolate and identify a causative pathogen. Subsequently, only PCL virus was positive, and qRT‐PCR was designed to detect the loading titre of PCL virus. We then initiated a heightened surveillance program on the pathogenicity and epidemiology of PCL virus. Results Six PCL virus strains, with severe diarrhoea and haemorrhagic enteritis, have been found in six different pig farms in Guangdong province, China. A multiple sequence alignment of these PCL viruses and bovine circovirus‐like virus/CH showed a similarity of 92.5‐94.8% for the Rep protein, indicating these PCL viruses are highly homologous to Bo‐Circo‐like virus associated with calf diarrhoea. There were striking similarities between the PCL virus and bovine circovirus‐like virus outbreaks in aetiological settings and Genomic sequence. We found that 11.2% (20/178) of diarrhoea samples and 13.3% (6/45) of pig farms were positive for PCL virus, suggesting that PCL virus may have spread widely in Pig farms. Moreover, this article underscores the risk of PCL virus spilling over and adapting to new species. Conclusions Porcine circovirus‐like virus was found to be associated with porcine diarrheal disease in China.

In addition to seven recognized CRESS DNA viral families, a great deal of novel CRESS DNA viruses needing to be classified by ICTV, and a group of viruses with large genomes (2833-3923 bp), according to genomic and Rep-phylogenetic characteristics, have been proposed family Kirkoviridae (Guo et al., 2018;Li et al., 2015;Shan et al., 2011).

Moreover, all the Rep proteins of the viruses belonging to proposed
family Kirkoviridae indicate significant genetic distance with those of the viruses within family Circoviridae (Guo et al., 2018;Shan et al., 2011;.The family Circovidae includes two genera: Cyclovirus and Circovirus. PCV-associated disease (PCVAD) is associated to PCV2 (porcine circovirus 2) infection (Meng, 2013), causing great harm to the pig industry. In recent years, Porcine-like virus P1, PCV3, and PCV4 have been reported to be found in pigs Wen et al., 2018).
In 2011, a novel pig-derived CRESS DNA virus was discovered in pig faces and named PCL virus (porcine circovirus-like virus) (Shan et al., 2011). A novel CRESS DNA virus named Bo-Circo-like virus from a calf with severe haemorrhagic enteritis has recently been detected in China (Guo et al., 2018), and three PCL viruses in pigs with diarrhoea have been found in Guangxi, China . The PCL virus belongs to proposed family Kirkoviridae, the prevalence and pathogenicity of which are rarely studied.
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 diarrhoea, enteritis, and vomiting. We obtained two blood samples, four faecal samples, and two intestinal samples from a pig farm suffered from diarrheal disease in the delivery room in September 2020 and attempted to isolate and identify a causative pathogen. Then, we identified microbial pathogens associated with diarrhoea, and only PCL virus was detected in the diarrhoea samples. Subsequently, we initiated a heightened surveillance program on the pathogenicity and epidemiology of PCL virus.
Five 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 (Supporting information Table S1). 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 (Takara). Whereas the ligands were transformed into DH-5α cells for gene cloning. The positive clones screened by PCR were sent to a commercial facility (Sangon Bioengineering Co., Ltd, China) for sequencing.

Real-time PCR array
To investigate tissue tropism of PCL virus in diarrheal piglets, a SYBR green quantitative real-time PCR (qRT-PCR) targeting the con-  Table S1). The results from three independent tests were analysed using GraphPad Prism 5.0 software.

Phylogenetic analysis
The complete gene sequences of PCL viruses obtained in this study have been uploaded to Genbank with the accession numbers (Supporting information Table S2). 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.

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 (Meng, 2013;Ouyang et al., 2019;Wen et al., 2018;Zhang et al., 2020). PCV2 have been associated with clinical diseases in pig farms known as PCV-associated disease (PCVAD), causing substantial economic losses (Meng, 2013). At present, PCV2 and PCV3 are widely popular in the global pig industry (Meng, 2013;Opriessnig et al., 2020). PCL virus is   Figure S1). Anatomy of diseased piglets found small intestine mucosa abscission, and intestinal mucosal lymph node enlargement (Supporting information Figure S1). There was a higher incidence of morbid-ity, while lower mortality among piglets as a result of relief of symptoms by breeder's saline rehydration used in piglets with diarrhoea.
Moreover, the PCL virus isolate CSW10 were detected in piglets aged 2 weeks at a farm in Shanwei (farm D), Guangdong province, and no other enteroviruses were detected in the samples (Supporting information    ures 3 and 4), indicating that all these viruses were most likely to belong to the same species. Fewer amino acid mutations on the Rep protein of these viruses were observed (Figure 4), the substitution effects on pathogenicity, replication, and infectivity require further investigation.
The striking similarities between the PCL virus and bovine circovirus-like virus outbreaks in aetiological settings and Genomic sequence cannot be ignored (Guo et al., 2018), suggesting that these strains may be the same virus and infect different hosts, also underscoring the risk of PCL virus spilling over and adapting to new species.
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.