Isolation and full-genome sequencing of porcine sapelovirus strain in piglets from Hainan province, China

Background: Porcine sapelovirus (PSV) is a species of the genus Sapelovirus within the family Picornaviridae, which associated with facute diarrhoea, respiratory distress, reproductive failure, and severe neurological disorders in swine. The first isolate strian of PSV was reported in Hainan province, China in 2019. Results: We report the isolation, genomic sequence of PSV isolated from pig diarrhea samples. The PSV strain was correctly identified by RT-PCR, IFA, WB assays, which appeared spherical with a diameter of approximately 25 nm by TEM. We named the strain PSV HaN01-CH2019, and its full genomes were 7,551 bp nucleotides in length. Phylogenetic analysis revealed that PSV HaN01-CH2019 and Chinese HuN01 strain are related in comparing with other reference strains. Conclusions: We successfully isolated the first PSV strain in Hainan province and prepared polyclonal antibodies. It is evident that PSV infection has occurred in Hainan province, and therefore, active molecular and serological investigation is important to swine population. Moreover, veterinarians must pay attention to this diarrhoea and reinforce biosecurity measures to prevent PSV spread.


Background
Porcine sapelovirus (PSV) that previously designated porcine enterovirus 8 in the genus Enterovirus is positive-sense single stranded RNA viruses within a family of Picornavirales, which is classified into the genus Sapelovirus belonging to the family Picornaviridae [1]. So far, the genus Sapelovirus consists of porcine sapelovirus, avian sapelovirus, simian sapelovirus and unclassified sapelovirus [1][2][3][4][5]. The full genome of PSV is approximately 7.5 kb nucleotides in length and share a similar genomic organization compared to other picornaviruses, which consists of a 5' terminal protein called VPg (Viral Protein genome-linked), a 5' untransated region (UTR), a large open reading frame (ORF), a 3' UTR and a poly(A) tail of variable length [6][7][8][9]. The single polyprotein encoded by a large ORF is subsequently cleaved into a leader (L) protein, four structural proteins (VP4, VP2, VP3 and   VP1) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D [7,10,11]. According to molecular and serological investigation revealed that PSV was highly prevalent in pigs around the world [5,8,[12][13][14]. PSV is a major pathogen worldwide, but there are few studies on the pathogenicity of PSV [5,13,15]. PSV has been reported to be associated with a wide spectrum of symptoms ranging from asymptomatic infection to clinical signs (including diarrhoea, polioencephalomyelitis, pneumonia, and reproductive disorders), and its infection in swine is most frequently asymptomatic and mainly transmits by a faecal-oral route [15,16].
Here, we describe the isolation of PSV from swine fecal samples from diarrhoea and biological characterization of PSV isolates. The PSV HaN01-CH2019 strain was characterized using an Immunofluorescence Assay (IFA) and Western Blot assay with a polyclonal antibody specific for VP1 of PSV capsid protein, RT-PCR assay with primer pair specific for the PSV 5'UTR region and transmission electron microscopy [17].

Methods
Fecal samples from a swine farm in Hainan province with diarrhea in May 2019. To determine etiology, fecal samples from diarrhoea were detected using TGEV, PEDV, PDCoV, PRV-A, PKV and PSV specific primers [17,18]. Total RNA of the specimens was extracted using the TRIzol reagent (Invitrogen, Grand Island, NY, USA) and 0.2 µg of total RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Vazyme, China) according to the manufacturer's instructions. PCR amplification was performed using various virus-specific primers (listed in Additional file 1: Table S1). Virus isolation and purification were carried out in a PK-15 cells culture system from the PSV-positive specimens and labeled PSV HaN01-CH2019. Rabbit anti-PSV VP1 polyclonal antibody was prepared in our lab using recombinant PSV VP1 protein. Identification of PSV HaN01-CH2019 using immunofluorescence assay (IFA), Western blot, and plaque assay was carried out as described previously [19]. Complete genome sequencing was performed using 7 pairs of overlapping primers (Additional file 1: Table S1)

Result
We successfully isolated PSV HaN01-CH2019 strain from piglets with diarrhoea, which was negative for PKV, TGEV, PEDV, PDCoV or PRV-A, but positive for PSV (Fig. 1). As shown in passages. The viral titer of fourth passage PSV HaN01-CH2019 strain was 7.2 × 10 8 PFU/mL. The plaque morphology was distinct and plaque size was approximately 1.5-2.5 mm in PK cells (Fig. 2b). To confirm the isolation of PSV, immunofluorescence assay and Western blot analysis were conducted using home-made rabbit polyclonal anti-PSV 5 VP1 antibody. As shown in Fig. 2c, cells infected by the isolate reacted with the specific rabbit polyclonal antibody against PSV VP1 protein by IFA. Meanwhile, Western blot analysis showed approximately 36 kilodalton (kDa) band in cells infected with the isolate (Fig. 2d, lane 2) but not in the mock cells (Fig. 2d, lane 1). TEM assay revealed icosahedral, nonenveloped viral particles with a diameter of approximately 25 nm (Fig. 2e).
These results indicated that PSV strain HaN01-CH2019 was successfully isolated.
The complete genome of PSV HaN01-CH2019 strain has been amplified, sequenced and  (Fig. 3a), and the polyprotein shares 92.9 ~ 95.2% amino acid sequence identity (Fig. 3b). Two phylogenetic tree results were constructed based on a complete genome sequence or ployprotein amino acid sequence and demonstrated that the PSV HaN01-CH2019 strain is closely related to PSV-HuN01 strain.

Discussion
PSV infection in piglets can induce diarrhoea and intestinal pathologys. Currently, the pathogenicity of PSV has been poorly studied [3]. In this study, a Chinese PSV strain isolated from Hainan province was reported for the first time. The PSV strain was identified by RT-PCR, IFA, WB and TEM assays. By comparing the homology of the whole genome sequence with the polyprotein amino acid sequence inferred in this study, it was found that PSV HaN01-CH2019 and the Chinese strain HuN01 were related to other reference strains.. Notably, epidemiological investigation revealed that the PSV-positive rates and co-infection of PSV/PEDV in diarrhea samples were much higher than in 6 asymptomatic samples, which indicating that PSV may contribute collectively to enteric disease of pigs along with other porcine pathogens [5,20]. Although PSV is an ancient virus, regard to its pathogenic and immunobiology has been poorly understood [21].

Availability of data and materials
The fecal samples from piglets with diarrhoea were kindly provided by a pig farmer in Hainan province (China) and stored in -80 °C. All primers used in this study were designed using Primer Premier 5.0 software. Data analyses were performed by the neighbor-joining (NJ) method with 1000 bootstrap r eplicates using MEGA 7.0 software.

Competing interests
The authors declare that they have no competing interests.