First Detection and Genetic Characterization of Equus Caballus Papillomavirus 1, 2 and 7 in China

Background: Nine species of Equus caballus papillomavirus (EcPV) have been reported to infect horses, however, there are so far no reports of such infections in China. Results: In our pioneer study with Chinese horses, we found EcPV-1 in intranasal papilloma and nasal swabs, EcPV-2 in nasal swabs and semen, and EcPV-7 primarily in semen. This indicates that EcPVs are indeed hosted by horses in China, and that EcPV-2 and 7 may be getting transmitted though breeding. Sequence analyses for complete genomic sequences of EcPV-1 (G2), EcPV-2 (XJ-KS1391) and EcPV-7 (XJ-zs1) were performed which indicated that EcPV-1, 2 and 7, that infect horses in China, share 99.3% nt identity with the already published sequences for EcPV-1, 2 and 7. These observations indicate that three types of EcPVs identied in the current study are highly similar variants of previously known types of EcPV-1, 2 and 7. Phylogenetic analysis based on L1 genes in GenBank showed that EcPV-1, 2 and 7, found in Chinese horses, are closely related to and clustered together with already known EcPV-1, 2 and 7, respectively. Conclusion: Our study provides a novel evidence for EcPVs infection and circulation in Chinese horses and thus lays the foundation for a systematic and detailed epidemiological study of these infections in Chinese horses.


Background
Papillomaviruses (PVs) are small, non-enveloped viruses harboring 8 kb of circular and double-stranded DNA genomes [1,2]. They are classi ed into different species of papillomaviruses according to the diversity of nucleotide sequences in the open reading frame (ORF) of the L1 gene [1,3,4]. The viruses infect different animals including humans, horses and bovine [1]. Till now nine species of EcPV, that are capable of infecting horses, have been documented [2,[4][5][6][7], however, there is no information on EcPV infection in horses within China.
In June 2018, we found papilloma-like tissue in the nose cavity of an eight years old thoroughbred stallion, of an equestrian club in Changji City, north-Xinjiang, China, that also experienced clinically manifested di culty in breathing. We designed this present study to perform metagenomic sequencing of the papilloma tissue from that stallion to characterize the virome of the papilloma and to identify the disease-causing pathogen.

cDNA library preparation
The cDNA library of the intranasal papilloma tissue was prepared according to a previously described method [8]. Brie y, the tissue was grinded and pooled to extract the viral nucleic acids that were reverse transcribed and then randomly ampli ed. The tagged and puri ed PCR products were sequenced at Shanghai Personal Biotechnology Co., Ltd. using one lane Illumina sequencing (HiSeq X-ten).

Pcr Analysis
We developed speci c PCR assay for each virus to con rm EcPV-1 in the next-generation sequencing results and to investigate the presence of EcPV2-9 in the horse population of north-Xinjiang. Primers were designed to amplify the various regions of the L1 gene for speci c and discriminative ampli cation of these viruses (Table S1). For ampli cation of the target genes we used TIANSeq HiFi Ampli cation Mix (Tiangen Biotechnology Co., Ltd) and used the following PCR program: 94°C for 2 minutes, 35 cycles of denaturation at 98°C for 10 seconds, annealing and extension at 68°C for 15 seconds and nal extension at 68°C for 5 minutes. All methods were performed in accordance with the relevant guidelines and regulations.

Different strains of EcPVs are present in a localized population of horses in China
Sequence analysis showed that a total of 61,077,096 sequence reads of 150 bp were generated from the tissue sample, of which 6,054 (0.0099%) could be annotated to known mammalian viruses (EcPV-1, Gemycircularvirus, Picobirnavirus, Murine leukemia virus, Human endogenous retrovirus W, etc). Among the various mammalian virus sequence reads obtained from the tissue sample, 1,690 reads shared more than 99.2% nucleotide identity with E6, E7 and L1 genes of EcPV-1 reference strains (GenBank accession nos. MF288893 and AF498323).

Discussion
Previous studies have shown that the EcPV-1 causes cutaneous papillomas in young horses, with age less than three years, that regress spontaneously within one to nine months [2,7,9]. However, as a novel nding we report EcPV-1 in the intranasal papilloma of an adult thoroughbred stallion (eight years-old) which unfortunately did not spontaneously return to health after 15 months. Even though it was treated by a veterinarian in November 2019, the horse could not progress with breeding due to breathing di culties persistent breathing di culties. In previous studies EcPV-1 has been shown to infect young horses with age as less as one to two years [9,10], however we were able to detect EcPV-1 in the nasal swabs of seven clinically healthy foals which were not even one year-old (Table 1).
It has been reported that EcPV-2 is present in the tissues of squamous cell carcinomas of sick horses and genitalia and conjunctiva swabs of healthy ones [11][12][13][14]. Our study is the rst of its kind to report EcPV-2 in nasal swabs and semen of Chinese horses (Table 1). Lange et al. were the rst ones to nd EcPV-7 in penile mass in horses in 2013 [4] and we also found EcPV-7 in horse semen (Table 1). However, the nasal swabs were devoid of any traces of EcPV-7. Detection of EcPV-2 and − 7 in semen suggests that the primary route for transmission of these two viruses could be through sexual activities in horses. The current study was performed in horses from Changji, Urumqi, and Yili indicating that EcPV-1, 2 and 7 are in circulation within different regions of north-Xinjiang in China.
The complete genomes of Chinese EcPV-1, 2 and 7 shared more than 99.3% nucleotide identity with previously submitted genome of EcPV-1, 2 and 7 to GenBank, respectively, indicating that EcPV-1, 2 and 7 had low genetic diversity. Homology analysis of the L1 genes indicated EcPV-1, 2 and 7 in Chinese horses possess high genetic identity.
According to EcPV taxonomy, that follows the general criteria established by the International Committee on the Taxonomy of Viruses (ICTV), an EcPV strain is recognized as a variant if the nucleotide sequence of the L1 ORF differs by less than 2% homology with the closest known EcPV type [1,3,4]. Based on this criterion, it is concluded that the EcPV-1, 2 and 7 strains in Chinese horses are indeed legit variants of referenced EcPV-1, 2 and 7 strains.

Conclusions
In our present study, we discover for the rst time three localized strains of EcPVs (EcPV-1, 2 and 7) in China and performed a thorough exploration of their basic genetic characteristics. This study is the rst to detect EcPV-1 in intranasal papilloma, EcPV-2 in nasal swabs and semen, and EcPV-7 in semen from localized Chinese population of horses. We demonstrate that the three EcPVs are indeed circulating among thoroughbred horses that have low diversities but different geographic distributions in north Xinjiang. This is also the rst report about the presence of EcPV-1, 2 and 7 in China, and thus a founding contributor towards any future molecular and sero-epidemiological and pathological studies for these viruses.
Declarations Figure 1 Phylogenetic analysis of EcPVs based on L1 genes. The black circles indicated the EcPV-1, -2 and -7 detected in this study.