First detection and complete genome analysis of porcine circovirus‐like virus P1 and porcine circovirus‐2 in yak in China

Abstract Porcine circovirus‐like virus P1, like porcine circovirus type 2 (PCV2), is a potential pathogen of post‐weaning multisystemic wasting syndrome in swine. Yaks are a valuable species and an iconic symbol of the Tibet Plateau which is the highest and largest plateau in the world. In this study, a total of 105 yak diarrheal samples, collected from 13 farms in Linzhi in the Tibet Plateau from January 2019 to December 2021, that were screened for P1 and PCV2 by polymerase chain reaction, 10.48% (n = 11) were positive for P1, 4.76% (n = 5) for PCV2, and 5.71% (n = 6) were positive for coinfection of P1 and PCV2. In addition, the whole genomes of eight P1 strains and eight PCV2 strains were sequenced. Alignment of deduced amino acid sequences of P1 ORF1 and PCV2 ORF2 gene revealed that ON012566 had one unique amino acid mutation at residues 137 (T to P). This mutation has important implication for the study of virus virulence, tissue tropism, and immune response. Phylogenetic analysis shows that the yak‐origin P1 strains in this study with cattle‐origin P1 reference strains were grouped into one cluster. The yak‐origin PCV2 (ON012566) and a buffalo‐origin PCV2 (KM116514) reference strain clustered in the same branch in the PCV2b regions. Meanwhile, the remaining PCV2 strains and buffalo‐origin PCV2 reference strain (ON012565) clustered in the PCV2d regions. To summarize, to our knowledge, this is the first report on the molecular prevalence and genome characteristics of P1 and PCV2 in yaks in the world and will contribute to further study of the molecular epidemiology, source, and evolution of P1 and PCV2 strains.


INTRODUCTION
Porcine circoviruses (PCVs) are small, non-enveloped DNA viruses and are members of circovirus genus of circovirus family (Mankertz et al., 2004). Generally, the PCVs genomes are single-stranded circular DNA, and the genome size varies from 1700 to 2000 bp. At present, PCV can be classified into four types: porcine circovirus type 1 (PCV1), PCV2, PCV3, and PCV4. PCV1 was originally discovered in 1974 and is considered to have no pathogenicity in pigs (Tischer et al., 1974). In 1996, a distinct virus was recovered from post-weaning multisystemic wasting syndrome (PMWS), and it is genetically and antigenically distinct from PCV1, named porcine circovirus 2 (Ellis et al., 1998). Recently, in the United States, PCV3 was identified in pigs with cardiac and multisystemic inflammation, and with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS) (Phan et al., 2016). intermediate (Mankertz et al., 1998;Nawagitgul et al., 2000). The clinical forms of PCV2 infection are PMWS, PDNS, porcine respiratory disease complex, reproductive failure, enteric, necrotizing lymphadenitis, exudative dermatitis, and congenital tremor (Allan & Ellis, 2000).
Therefore, the diseases associated with PCV2 infection have a serious economic impact on the swine industry worldwide.
Porcine circovirus-like virus P1 is an emerging causative pathogen of PMWS . The genome of P1 consists of a covalently closed, single-stranded, circular molecule of only 648 nucleotides which is highly homologous to the partial genome sequences of PCV2 (AF381175), but different in only 16 consecutive nucleotides (CGTTACTAGTGGATCC) (D. Zhang et al., 2018). P1 shares 64.39% identity with the consensus region of PCV1 (GU722334) . The P1 genome encodes three major ORFs: ORF1 and ORF2 are encoded on the antisense strand, whereas ORF3 is encoded on the sense strand. P1 ORF1 encodes the 27.8-kDa viral capsid protein (Cap) and has extensive homology to the N-terminal domain of the PCV2 Cap protein (Wen et al., 2014). Like PCV2, P1 can reproduce many clinical symptoms, such as pallor of the skin and diarrhoea . In addition, P1 has been prevalent in China since its discovery .
Recently, the P1 virus was found in various animals, such as dogs, pigs, cattle, cats, rabbits, and goats (Wen, Mao, Fan, et al., 2017;Wen et al., 2020). In 2007, the presence of PCV1 in wild boars in Germany was described using serological methods (Cságola et al., 2008). Moreover, in 1995, for the first time, Tischer et al. (1995) confirmed the presence of PCV1 antibodies in cattle in Germany. For PCV2 in nonporcine hosts, there was evidence in previous studies. In Canada, a PCV2 nucleotide was identified in cattle with respiratory diseases and aborted bovine fetuses (Nayar et al., 1999). In the United States and China, PCV2 was frequently detected in beef from supermarkets, beef stalls, and goat samples (Li et al., 2011;Zhai et al., 2016;W. Zhang et al., 2014). In addition, PCV2 was suggested as a potential causal factor of haemorrhagic diathesis, which can cause moderate clinical signs, viraemia, and seroconversion after infecting calves (Halami et al., 2013(Halami et al., , 2014Kappe et al., 2010). Stunning, PCV1, and PCV2 were detected in human samples, respectively (Bernstein et al., 2003;Esona et al., 2014;Li et al., 2010). It can pose a significant threat to public health and security.
The yak (Bos grunniens) belongs to the genus Bovidae and is a unique species of long-haired cattle, with a population of about 15 million yaks living on the Tibetan plateau over 4000 m above the sea level, accounting for more than 90% of the total yak population in the world (Cui et al., 2020;Yang et al., 2017;Q. Zhang et al., 2016). Yak has high adaptability to harsh environments of high elevation, strong ultraviolet, low oxygen, and low temperatures, and is capable of providing meat, milk, skins, transport, and fuel (faeces), making it an indispensable animal for the local people in the Tibet Plateau (Wu et al., 2020).
However, diarrhoea in yak is a common disease, and many viruses such as bovine viral diarrhoea virus and Nebovirus have been identified as important diarrhoea-causing viruses circulating in the yak. Interestingly, P1 or PCV2 can cause diarrhoea in swine (Allan & Ellis, 2000;. However, the information regarding the prevalence characteristics of P1 or PCV2 in yaks remains unclear. Here, we aimed to investigate the prevalence and molecular characteristics of P1 or PCV2 in yaks in Linzhi, Tibet. To the best of our knowledge, this is the first report of the circulation of P1 and PCV2 among yaks in the world. 2) were used for sequence alignment and mutation analysis. We used 28 representative strains (P1, n = 13; PCV2, n = 15) for phylogenetic analysis, including cattle-origin P1 strains, rabbit-origin P1 strains, goat-origin P1 strains, pig-origin P1 strains, buffalo-origin PCV2 strains, pig-origin PCV2 strains, and human-origin PCV2 strains.
Moreover, PCV3 and PCV4 were also detected in these samples (data not shown). We detected P1 and PCV2 in yak diarrhoeic samples for the first time, which provides the possibility that P1 and PCV2 can be transmitted through yak faeces and have important implications for further investigation of whether P1 and PCV2 cause diarrhoea in yaks.
In previous studies, there was a risk of cross-species transmission of P1 or PCV2. In addition, yaks are free-grazing domestic animals in the Tibet Plateau, with a large living area where Tibetan pigs and many wild ruminants are found. Therefore, in these regions, P1 or PCV2 has a risk of cross-species transmission that will bring great challenges to virus prevention and control.
To further study the molecular epidemiology of P1 and PCV2, eight complete yak-origin P1 and eight complete yak-origin PCV2 sequences were obtained from P1-positive and PCV2-positive clinical samples detected in this study and submitted to the GenBank database (the host, size, genotype, and GenBank accession numbers of these strains are summarized in Table 1 -87, 113-139, and 193-207) (Morozov et al., 1998). Change in amino acid composition at these sites may alter virus characters such as virulence, tissue tropism, and immune response (Saha et al., 2011). The strain ON012566 had one difference at position 137 (T to P) from other reference PCV2-ORF2 amino acid sequences ( Figure 3).
Therefore, further study on this special site will be interesting.
A corresponding phylogenetic tree (including 13 reference P1 sequences and eight P1 sequences from this study) was con- Interestingly, all P1 sequences in this study are to be clustered in a large branch of the phylogenetic tree and similar with two cattleorigin reference strains (KY462784 and KY462783), and the eight P1 sequences were closely related to each other (Figure 4)

F I G U R E 2
Analysis of nucleic acid sequence and amino acid sequence of P1 ORF1. The nucleotide sequence of P1 ORF1 (a) and the deduced amino acid sequence (b) were compared with the previously reported sequences and amino acid sequences of P1 ORF1 genes, respectively. This alignment included deduced nucleotide sequences of 8 P1 ORF1 sequences from this study and 13 P1 ORF1 reference sequences from Genbank. All the sequences were aligned in Clustal W and viewed in BioEdit sequence alignment editor tool. Mutant strains and mutant sites are marked with red underlines.

F I G U R E 3
Alignment of deduced amino acid sequences of porcine circovirus type 2 (PCV2) ORF2 gene.This alignment included deduced amino acid sequences of ORF2 from 8 PCV2 sequences from this study and 15 PCV2 reference sequences of different genotypes from Genbank. All the sequences were aligned in Clustal W and viewed in BioEdit sequence alignment editor tool. Mutation sites marked with red underline or red box. Position 89, marked with a blue box, could differentiate PCV2a, PCV2b, and PCV2d, which harbored isoleucine (I), arginine (R), and leucine (L) residues, respectively.
These results provide a basis for structural and functional studies of the P1 and PCV2.
The Tibet Plateau, with an average altitude of 4000 m, thin air, strong ultraviolet radiation, and large temperature difference throughout the year, has bred many special species, including yaks and Tibetan pigs, and endowed these species with strong tolerance and cold resistance (Ma et al., 2019;Wu et al., 2020;Yang et al., 2017). Research on the genetic evolution of the virus in such a special environment is of great significance. In previous studies, there is a risk of cross-species transmission of P1 or PCV2 (Esona et al., 2014;Wen, Mao, Fan, et al., 2017). In this research, genetic phylogenetic tree analysis showed that yak-origin P1 was located in several branches and distributed relatively intensively, indicating that yak-origin P1 had a certain regional specificity and genetic diversity. In addition, the phylogenetic tree analysis showed that the yak-origin P1 was closest to P1 isolates from cattle-origin and far away from domestic pigs, rabbits, goats, and other species, indicating that the yak-origin P1 virus had a certain species specificity. In previous studies, PCV2-infected calves demonstrate that host susceptibility of PCV2 is not solely restricted to pigs (Halami et al., 2014). In this study, the phylogenetic tree analysis showed the yak-origin PCV2 strains were in separate branches and far away from domestic pigs and humans, indicating that the yak-origin PCV2 has certain species specificity and genetic diversity ( Figure 5) In conclusion, to our knowledge, this is the first report on the molecular prevalence and genome characteristics of P1 and PCV2 in yaks in the world. This report investigated the circulation of P1 or PCV2 in yaks in China, showing genetic diversity. However, further studies will be required to ascertain whether P1 or PCV2 causes diarrhoea in yaks.
This finding will contribute to the diagnosis and prevention of yak diarrhoea in China, and it will contribute to further study of the molecular epidemiology, source, and evolution of P1 and PCV2 strains in yaks.

F I G U R E 5
Phylogenetic analysis of eight yak-origin porcine circovirus type 2 (PCV2) strains in this study and other reference strains. The phylogenetic tree was constructed by the neighbor-joining method using MEGA 11.0 software. Bootstrap replications were set by 1000. Note: Eight yak-origin PCV2 strains in this study labeled by blue circles and two buffalo-origin PCV2 reference sequences were labeled using underlines.

ACKNOWLEDGEMENT
We thank Wenjie Yuan (Nanjing Agricultural University) for assistance with data visualization.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The genome sequences resulting from this study were deposited into the GenBank database with accession numbers MW263905, ON012558-ON012566, and ON793631-ON793636.

ETHICS STATEMENT
The study did not involve animal experiments, and there were no animal ethics problems.