A number of viruses have been discovered that can cause acute diarrhea in China’s piglets, which has resulted in massive losses to the pig husbandry [7, 9]. A number of previous studies have been showed that kirV can exhibit genetic diversity in both horse and donkey [2, 3], hence to investigate the presence of kirV in swine, DNA was extracted from the collected anal swab samples. After performing PCR with the primer pairs, Hp4-F and Hp4-R, as well as agarose gel electrophoresis, kirV was detected in 150 of 410 rectal swabs of piglets negative for the major diarrhea viruses (PEDV, TGEV, PRV-A, and PDCoV), thus indicating that this virus can infect piglets. Our results suggested that kirV can infect a wide range of host as the virus was also detected in sheep and calf (data not shown). However, further studies are needed to investigate the infection of kirV in other livestock and to confirm host range of this virus.
Interestingly, among 150 kirV-positive rectal swabs, 149 kirVs were detected in all diarrhoeic samples, however, one of 261 healthy samples was positive for this virus. Therefore, based on the results, it can be concluded that the kirV infection was strongly association with diarrhea of piglet, Xinjiang, China. Until now, several viruses, like PEDV, etc., have been reported to cause severe damage to the development of the pig industry, thus causing major economic losses to global porcine farms [7, 9]. In the current study, we have established that kirV could be a potential novel diarrhea virus, but this aspect needs to be investigated on porcine population of worldwide. Thus, the future studies should aim to isolate the kirV, then perform challenge assay and confirm the association between the virus and piglet diarrhea.
In addition, as reported for horse and donkey kirVs in two previous studies [2, 3], some variations in Hp4 genes of piglet kirVs were also observed. However, the HP4 sequences from all piglet kirVs in this study shared 99.6–100% nt identity, thereby indicating the genetic conservation. Indeed, piglet and donkey kirV Hp4 genes displayed high 99.2–99.5% nt identity, which suggested that the kirVs of piglet and donkey could have common evolutionary origins.
Interestingly, several prior studies have showed that kirV had relatively larger genomes [1–3]. Among these viruses, the complete genome sequences of horse kirV are 3,732, and 3,800 nt, and donkey kirV is composed of 3,750 nt. Thereafter, to facilitate understanding of the genetic characterization of piglet kirV, all piglet kirV Hp4 genes sequences were aligned and one primer pair, Com-F and Com-R, were designed based on the Hp4 gene conservative region (Table S1). After agarose gel electrophoresis, the PCR products amplified by the primer pairs displayed bands of 3,410 bp. After sequencing and assembly with Hp4 gene identified as belonging to donkey kirVs, three 3,750-nt complete genome sequences were obtained (named Cj-D5, Cj-D32, and Cj-D43) (GenBank nos. MW504209–MW504211), with 12 nt longer than kirV Cj-7-7 and 50 nt shorter than that of kirV Equ1. It shared 86.5–89.0% homology with horse kirV Cj-7-7 as well as Equ1, and shared 99.9% homology with donkey kirV Hetian-46, Hetian-48 and Hetian-58. Therefore, based on these findings, it could be inferred that piglet, and donkey kirV had common evolutionary origins. Similar to the complete genomic map of horse and donkey kirVs, porcine kirV possessed two forward reading frame (nt 1–918, and nt 1605–1991) and four reverse reading frames (nt 3629–3156, nt 3133–2567, nt 2544–1973, and nt 1940–1350) (Fig. 1). Among various identified reading frames, forward reading frame (nt 1–918) and reverse reading frames (nt 3133–2567) encoded the replicase protein (Rep, 305 amino acid (aa)) and the putative capsid protein (Cap, 188 aa), respectively. The other four ORFs encoded four hypothetical proteins (Hp) of relatively unknown function with sequences of 196, 190, 157 and 128 aa, respectively (Fig. 1).
In addition, a previously published article has already revealed that circovirus and cyclovirus Rep have six well-conserved RCR and SF3 helicase motifs, including motif I [FT(L/I)N], motif II [PHLQG], motif III [YC(S/x)K], Walker-A [G(P/x)(P/x)GxGK(S/t)], Walker-B [uuDDF], and motif C [uTSN] where ‘‘x’’ represents any residue, and ‘‘u’’represents a hydrophobic amino acid (F, I, L, V, M) [10–12].
It was noted that similar but different to Rep of the members of the family Circoviridae, Rep of piglet kirVs identified in this study, as well as that of donkey and horse kirVs reported in in previous three studies [1–3] all had conserved motifs RCR and SF3 helicase motifs, including RCR motif I [FTIN], motif II [PHIQG] and motif III [YCSK] and dNTP-binding or P-loop NTPase domains characteristic of the superfamily 3 (SF3) helicases motifs Walker-A [GPAGVGKS], Walker-B [RVVLDD], and motif C [VTSN]. But kirV had more complex genomic structure (six ORFs, ORF > 300 nt) in comparison to circovirus and cyclovirus (Fig. 1). Thus, based on our findings kirV could be potentially classified into a new family: Kirkoviridae.
Moreover, identical to circovirus and cyclovirus conserved origin of replication (ori) “TAGTATTAC” [10–12], kirV ori of different host was characterized by a conserved nonanucleotide motif “CAATATTAC” (Fig. 1). These results suggested that the kirV could have a similar replication strategy as that of the members of the family Circoviridae.
A comparison of the Rep genes revealed that three distinct kirV identified in our study shared 99.9–100% nt identity, and 99.7–100% aa similarity, thus indicating that the piglet kirV possessed a high genetic identity. Further multiple sequence alignment of three piglet kirV identified in the present study revealed a sequence similarity of 44.3–99.9%, 37–41.8%, 40–43.8% for the Rep gene sequence, and a sequence similarity of 32.9–99.7%, 24.1–40.1%, 23–25.9% for the Rep protein sequence after taking into account the sequences of available reference strains of the proposed family Kirkoviridae, genus Circovirus and genus Cyclovirus, respectively.
A phylogenetic tree was then reconstructed using the maximum likelihood method based on the Rep protein sequences to understand the potential genetic relationships between piglet kirV and available reference strains. The results indicated that piglet, donkey, and horse kirVs formed an independent branch, and displayed most close relationship to donkey kirV Hetian-58, Hetian-48, and Hetian-47, as indicated by both nt and amino acid homology analyses. These kirV and human, rodent, horse, pig, bovine and macaca mulatta-origin CRESS DNA viruses were clustered into the proposed family Kirkoviridae but was divergent from the lineages of Circovirus, and Cyclovirus (Fig. 2). Unfortunately, similar to previous studies [2, 3], we also attempted to isolate the kirV using PK15, ST and Vero cells, but were not successful. Next study will continue to try to isolate viruses using other types of cells.
Overall, this study provides the first molecular evidence for existence of piglet kirV, and our observations indicate that the virus may function as a novel porcine diarrheic associated virus. however, further research is required to develop innovative methods for virus isolation and to explore pathogenesis mechanisms associated with this virus.