Several bacteria and viruses are known to cause gastrointestinal infections in piglets leading to huge economic losses. Recently, a relatively new virus, the porcine kobuvirus was found to be associated with swine gastroenteritis [1]. Porcine kobuviruses are small, non-enveloped viruses with single-stranded, positive-sense genomic RNA within the family Picornaviridae, genus kobuvirus [2]. The virus was first detected in 2008 in fecal samples of domestic pigs in Hungary. The complete genome of the S-1-HUN strain is 8,210 nucleotides in length, including a leader (L) protein, three structural (VP0, VP3, and VP1), and seven non-structural (2A, 2B, and 2C; and 3A, 3B, 3C, and 3D) proteins [3]. In this study, we report for the first time the detection of porcine kobuvirus in fecal samples of diarrheic and healthy pigs in U.S. swine farms. Phylogenetic relationship of these strains with the reference strains of kobuviruses is also presented. Intestinal contents from 114 diarrheic pigs submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) for disease diagnosis were included in this study to determine the presence of porcine kobuvirus. Before screening for kobuvirus, the samples from diarrheic pigs had been tested at the MVDL for the presence of common intestinal pathogens including TGEV (transmissible gastroenteritis virus) and rotavirus groups A, B, and C using quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) [4]. In addition, fecal samples from 46 apparently healthy pigs from three different farms were also tested.

Viral RNA was extracted from samples using the QIAamp viral RNA easy kit (Qiagen, Valencia, CA). The reverse transcription-polymerase chain reaction (RT-PCR) was done using Qiagen One Step RT-PCR kit (Qiagen) and universal kobuvirus primers of 3D (RNA-dependent RNA polymerase) and VP1 region (capsid protein) [3, 5]. The PCR products were analyzed in 1.2 % agarose gels containing 0.5 μg/mL ethidium bromide and then visualized by ultraviolet illumination. Amplicons of 216 and 811 bp were expected. Of the 114 specimens from ill pigs, 25 were positive for porcine kobuvirus with 3D gene primers. The highest positivity rate of 68 % (p < 0.05) was observed in piglets under the age of 4 weeks followed by 20 % in pigs up to 8 weeks and 4 % in pigs above 9 weeks. All 25 specimens showed mixed infection with TGEV (8 %) or with rotavirus (72 %). Of the 46 fecal samples from healthy pigs, 10 were found positive.

PCR products were purified using a commercial PCR purification kit (Qiagen) and sequenced at the ACGT (Wheeling, IL). Sequencing was conducted in both directions using the same primers as used in RT-PCR. Forward and reverse sequences were aligned together using Sequencher software (http://genecodes.com/). The obtained sequences were compared with the reference strains available in GenBank and aligned using CLUSTAL W software. Phylogenetic analysis was conducted using MEGA version 5.0 [6]. Neighbor-joining trees were constructed using the Kimura 2 parameter model, and the reliability of different phylogenetic groupings was evaluated by the bootstrap method with 1,000 replications (Fig. 1a, b).

Fig. 1
figure 1

Phylogenetic trees of porcine kobuvirus strains constructed based on a partial nucleotide sequences of 3D region (208 bp), b VP1 region (780 bp). Our strains are underlined and identified by abbreviations—specimen number, year of illness, and name of the country. Scale indicates genetic distance

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Partial nucleotide sequences of 3D region of 25 porcine kobuvirus strains revealed 93.1–96.5 % nucleotide identity with NLD 45 strain from the Netherlands and BRA24 strain from Brazil. The 10 strains from healthy pigs also showed similar nucleotide identity (data not shown). The 25 strains from diarrheic pigs were further typed using primers from VP1 region (capsid protein). Only 10 of 25 (40 %) were typeable with VP1 region primers. In the VP1 region, strains from diarrheic pigs showed only 86.7–88.5 % homology with the T247 strain from Japan and 85.8–87.4 % homology with WUH1 strain from China. The sequences of the porcine kobuvirus strains were submitted to GenBank under the following accession numbers: from JX543949 to JX543973 for 3D region sequences and from JX987498 to JX987507 for VP1 region sequences of strains from diarrheic pigs. The accession numbers for sequences of 3D region of strains from healthy pigs are from KC139253 to KC139262.

Porcine kobuvirus has previously been detected in both healthy and diarrheic pigs albeit at different rates. For example, kobuvirus was reported in 19.3 % of fecal samples from pigs without diarrhea and 84.5 % from pigs with diarrhea in 2010, and 45.5 and 32.6 % in non-diarrheal and diarrheal samples, respectively, in 2011 from Korea. In Japan, the detection rate of kobuvirus in healthy young pigs was 45 % [7, 8]. In a study in Thailand, 99 % of fecal samples collected from diarrheic pigs were reported as kobuvirus positive [9]. In the present study, the prevalence of kobuvirus was very similar in healthy (21.7 %) and ill (21.9 %) pigs. These rates are much lower than those observed in Thailand and Korea. However, high positivity rate of kobuvirus infection in piglets under the age of 4 weeks in our study is similar to that reported from Shanghai, China [10]. Our results showed, all 25 kobuvirus positive pigs had co-infections with other enteric viral pathogens, e.g., rotavirus and/or TGEV. Also, samples from 11 of the 15 states were positive suggesting that kobuvirus strains are co-circulating in many different U.S. states.

The role of porcine kobuvirus currently is unknown because of its presence in both healthy and ill piglets. Some of the possibilities are that (i) there exist two different pathotypes of porcine kobuvirus namely, pathogenic and non-pathogenic; (ii) the virus load is higher in ill pigs versus healthy ones; (iii) kobuvirus causes diarrhea only in the presence of other pathogens such as rotavirus or other enteric viruses; and (iv) kobuvirus is an endogenous passenger virus and is of no consequence. Nonetheless, our preliminary results indicate the need for further studies on transmission pattern and pathogenesis of porcine kobuvirus to determine its etiological role as a viral pathogen in the U.S. swine. Studies are also needed to characterize the circulating strains and their involvement in swine diarrhea. Since kobu-like viruses have been isolated from humans, it is also important to study the zoonotic potential of porcine kobuviruses.