Histopathology of Porcine kobuvirus in Chinese piglets

  • Fan Yang,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Xiaowan Liu,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Yuancheng Zhou,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Wenting Lyu,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Siyao Xu,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Zhiwen Xu,

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

  • Ling Zhu

    abtczl72@126.com

    Affiliation Animal Biotechnology Center, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611134, China

    0000-0002-0355-3044

Histopathology of Porcine kobuvirus in Chinese piglets

  • Fan Yang, 
  • Xiaowan Liu, 
  • Yuancheng Zhou, 
  • Wenting Lyu, 
  • Siyao Xu, 
  • Zhiwen Xu, 
  • Ling Zhu
x

Dear Editor,

Kobuvirus, classified as a new genus within the Picornaviridae family in 1999, is a non-enveloped virus with single-str and ed, positive-sense genomic RNA(Pringle, 1999). This genus contains 3 species currently recoganized: Aichivirus A(AiV), Aichivirus B(BKoV), and Aichivirus C(PKoV). PKoV was first detected in fecal samples from clinically healthy domestic pigs in Eastern Hungary in early 2007(Reuter et al., 2008) and since, continually reported among domestic pigs in different countries, including China(Yu et al., 2009). In 2010, Wang et al. screened 116 stool specimens, collected from 3 pig farms in Shanghai and detected PKoV infection rates of 46.7%, 35.1%, and 32.4%(Wang et al., 2011). Additionally, a complete genome sequence analysis for PKoV and evolution of PKoV infection have been previously reported(Reuter et al., 2009; Reuter et al., 2010). However, few studies focused on the clinical manifestations and histopathology of piglets infected by PKoV. The aim of this study was to determine whether PKoV caused specific histopathological lesions in infected piglets.

A fecal sample each, was collected from a total of 12, 10-day-old piglets, which showed clinical manifestations of PKoV, including diarrhea, emaciation, and nausea at a large-scale hog-farm in Ya'an in November 2012. Additionally, samples of liver, lung, and stomach tissue, as well as intestinal contents were collected from 2 of the 12 piglets, for isolation and identification of bacteria and virus. Bacteria were isolated by aerobic and anaerobic culture at 37 ℃ for 48 h using blood agar plates(5% defibrinated sheep blood). The results showed that the tissue samples were negative for common pathogenic bacteria, including Listeria, Leptospira, Mycoplasma, Brucella species, pathogenic Escherichia coli, Salmonella, Pasteurella, Haemophilus parasuis, Streptococcus hemolyticus, Campylobacter jejuni, and Clostridium perfringens type C. Viruses were identified by polymerase chain reaction(PCR)or reverse transcriptase(RT)-PCR using specific primers(Supplementary Table S1). Total DNA and total RNA were extracted using NucleoSpin Tissue Kit(Macherey-Nagel GmbH, Germany), and Trizol(Invitrogen Corp, Carlsbad, CA, USA), respectively. The amplification products were analyzed using 1% agarose gel electrophoresis. The results showed that fecal, serum, heart, liver, spleen, lung and kidney samples were positive for PkoV, but negative for 16 other tested viruses, including porcine pseudorabies virus(PRV), porcine parvovirus(PPV), porcine bocavirus(PBoV), porcine circovirus type 2(PCV2), classic swine fever virus(CSFV), group A rotavirus(GARV), group B rotavirus(GBRV), group C rotavirus(GCRV), porcine sapovirus(PSaV), porcine norovirus(PNoV), porcine torovirus(PToV), Japanese encephalitis virus(JEV), porcine epidemic diarrhea virus(PEDV), porcine reproductive respiratory syndrome virus(PRRSV), transmissible gastroenteritis virus(TGEV), and bovine viral diarrhea virus(BVDV).

Different samples from the 2 piglets were homogenized to obtain 10%(w/v)suspensions in stroke-physiological saline solution(pH 7.2-7.4), and filtered through 0.22 μm syringe filters. Subsequently, filtrates(1 mL)were added to Vero cells and incubated for 1 h at 37 ℃, after which, the mixtures were transferred to flasks containing 5 mL Dulbecco's Modified Eagle's Medium(DMEM)supplemented with 2% fetal bovine serum. After up to 5-10 blind passages, cytopathic effect(CPE)was not observed, but RT-PCR results were positive for PKoV.

Using plaque purification and electron microscopy, 2 cell-adapted Kobuvirus strains were obtained, of which one PKoV strain was selected and administered to piglets in this study(GenBank Number: JQ724539.1). A total of 20, 10-day-old, specific pathogen-free piglets were r and omly divided into 4 groups, of which, 3 were intramuscularly administered 2 mL PKoV virus(107 PFU/mL), while the control group was intramuscularly administered 2 mL DMEM(Gibco, USA). At 2, 4, 6, 8, and 10 days post inoculation(dpi), 1 piglet from each of the 4 groups was euthanized. The control group showed no clinical manifestations throughout the study, with no obvious gross lesions. On the other h and, piglets injected with PKoV developed obvious clinical manifestations such as diarrhea, emaciation, and nausea at 4 dpi, while 2 piglets died from severe olighydria at 6 dpi. Clinical manifestations in the surviving piglets began to disappear after 8 dpi. Necropsy revealed petechial hemorrhage on the surface of the kidneys, while milk agglutination and flatulence was observed at 4 and 6 dpi(Supplementary Table S2). Tissue samples collected from each of the 20 piglets(PCR detection results are shown in Figure 1)were fixed in 10% neutral-buffered formalin for 24-48 hours, followed by routine processing and embedded in paraffin wax. Sections were stained with hematoxylin and eosin(HE) and observed under a light microscope.

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Fig 1. (A) PCR results of mixed piglet tissue samples (lung, kidney and digestive system). M: DNA Marker DL 2000; +: Positive sample; -: Negative sample; Lanes 1 to 4, 5 to 8, 9 to 12, 13 to 16, 17 to 20: One piglet each, from the control group and 1st, 2nd and 3rd experimental group euthanized at 2, 4, 6, 8, and 10 days post inoculation (dpi); Lanes 10, 11: two piglets that died at 6 dpi. (B-G) Histological lesions in multiple organs (HE staining). Original magnification: 400× (B-1, B-2, C-1, C-2, D-1, D-2, E-2, E-3, E-4, E-5, G-2), 200× (E-1, F-2, F-3, G-1), and 100× (F-1, B-3). B-1 and B-2: Examination of the lungs from the 2 pigs that deceased at 6 dpi revealed a large quantity of defluvium bronchial epithelial cells in the pulmonary artery, with a few defluvium alveolar epithelial cells exuded to the alveoli, and the presence of lymph node-like cells, while the pulmonary interstitial tissue appeared to be thickened and congested; B-3: Lung tissue form the control group did not show any obvious changes; C-1, C-2: Kidney samples from the 2 pigs that deceased at 6 dpi showed cellular cast formation with a large number of red blood cells, and several renal tubular epithelial cells exuded in the kidney tubules; D-1 and D-2: The digestive system of the piglets that experienced diarrhea showed marked extravasated blood from the stomach, with few lymphocytes and mononuclear phagocytes infiltrating the submucosa; E-1 and E-2: The duodenum of the piglets that experienced diarrhea showed defluxion of a few epithelial cells of the villi intestinalis as well as a number of inflammatory cells, especially mononuclear lymphocytes and neutrophils, infiltrating the lamina propria of the duodenum; E-3: For the piglets that experienced diarrhea, blood vessels of the lamina propria were congested with red blood cells; E-4: For the piglets that experienced diarrhea, increased proliferation of goblet cells was observed in the villi intestinalis, with sections of villi intestinalis defluxion; E-5: The duodenum of the control group did not show any obvious changes; F-1, F-2: Numerous goblet cells were found in the villi intestinalis of the cecum at 6 dpi; F-3: The cecum of the control group did not show any obvious changes; G-1 and G-2: At 10 dpi, a large number of lymphocytes had infiltrated the submucosa of the rectum.

Examination of the lungs from the 2 pigs that deceased at 6 dpi revealed a large quantity of defluvium bronchial epithelial cells in the pulmonary artery, with a few defluvium alveolar epithelial cells exuded to the alveoli and the presence of lymph node-like cells, while the pulmonary interstitial tissue appeared to be thickened and congested, suggesting interstitial pneumonia(Figure 1B-1, 1B-2). The kidney showed cellular cast formation, with a large number of red blood cells, and several renal tubular epithelial cells exuded in the kidney tubules, suggesting kidney petechial hemorrhage(Figure 1C-1, 1C-2). The digestive system of the piglets that experienced diarrhea showed marked extravasated blood from the stomach, with few lymphocytes and mononuclear phagocytes infiltrating the submucosa(Figure 1D-1, 1D-2); the duodenum showed defluxion of a few epithelial cells of the villi intestinalis as well as a number of inflammatory cells, especially mononuclear lymphocytes and neutrophils, infiltrating the lamina propria of the duodenum(Figure 1E-1, 1E-2); blood vessels of the lamina propria were congested with red blood cells(Figure 1E-3); increased proliferation of goblet cells in the villi intestinalis, with sections of villi intestinalis defluxion(Figure 1E-4). At 6 dpi, numerous goblet cells were found in the villi intestinalis of the cecum(Figure 1F-1, 1F-2) and at 10 dpi, a large quantity of lymphocytes had infiltrated the submucosa of the rectum(Figure 1G-1, 1G-2). The control group did not show any obvious changes(Figure 1B-3, 1E-5, 1F-3). From the finding of inflammatory cells in the stomach, duodenum, and rectum, we conclude that PKoV induces an inflammatory response and thus, may play a role in pig diarrhea and cause gastroenteritis, similar to a previous report(Park et al., 2010).

In this study, our main purpose was to determine whether PKoV causes histopathological lesions after infection of piglets. PCR results confirmed that the piglets were indeed infected by PKoV. Additionally, we chose healthy experimental animals in this study, reared under st and ard conditions, such as feed and management, therefore, we speculated that pathological changes observed were caused by PKoV. To date, no studies have reported that PKoV causes CPE in cell culture, as seen in this study. We in fact, obtained 2 cell-adapted Kobuvirus strains by plaque purification and electron microscopy, which indicates cell compatibility. This could also be related to environmental factors and viral mutations. In conclusion, we showed that PKoV, as a unique causative agent, produced diarrhea and specific pathological lesions, which may lead to interstitial pneumonia, nephrosis, and gastroenteritis. This study provides an important basis for further research on the pathogenic characteristics of PKoV. Moreover, further studies are required to obtain direct evidence for pathogenesis, transmission, immunology, and epidemiology of PKoV.

FOOTNOTES

This study was supported by Changjiang Scholars and Innovative Research Team in University(Project No: IRT13083), Sichuan Youth Waterfowl Disease Control and Prevention Science and Technology Innovative Research Team(Project No: 2013TD0015) and Sichuan Province Science and Technology Support Program(Project No: 2014NZ0043). The authors declare that they have no conflict of interest. All the animal tests comply with Sichuan province laboratory animal management approach and the requirement of animal welfare. Piglet samples for the study were collected with the farm owner's approval, and the experiments were conducted under the guidelines of the University of Sichuan Agricultural for animal experiments.

Supplementary tables are available on the website of Virologica Sinica: www.virosin.org; link.springer.com/journal/12250.

Electronic Supplementary Material

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Table S1. List of oligonucleotide primers designed for detection and sequencing.

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Table S2. The lesions of those infected piglets.

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