Introduction

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that can cause enteric disease characterized by watery diarrhea, vomiting, dehydration, and growth retardation. The mortality rate is about 40%-80% in nursing piglets [1]. PDCoV is an enveloped positive-sense single-stranded RNA virus that belongs to the genus Deltacoronavirus within the family Coronaviridae [2]. The size of PDCoV genome is about 25.4 kb, the smallest genome among the known coronavirus. The genome encodes two large polyproteins (ORF1a and ORF1b), four structure proteins (spike, membrane, envelope, and nucleocapsid), and two accessory proteins (NS6 and NS7) [3, 4]. The spike (S) protein is the key responsible for virus entry. It also functions as the main antigen for the induction of protective antibodies. In addition, the S gene is the primary gene used for studying the genetic diversity of coronavirus isolates.

PDCoV was first reported in Hong Kong in 2012 [5]. Later, it caused outbreaks in the United States in 2014 [6]. Since then, the virus has spread to various countries, including Canada [7], South Korea [8], Japan [9], Vietnam [10], and Thailand [11, 12], causing tremendous financial losses to the pork industry.

In China, the emergence of PDCoV was first reported in 2015 [13, 14]. Several PDCoV strains have been isolated in northern, southern, and central China [3A). To further characterize the putative recombination events, we performed nucleotide similarity comparisons between strain BN23 and other PDCoV strains, using SimPlot v.3.5.1. This analysis confirmed the BN23 was a recombinant strain with the recombination breakpoints map** to ORF1a (Fig. 3B). These results suggest that BN23 was generated by a natural recombination event between the Thailand/S5015L/2015 and CHN-AH-2004 strains (Tables 3, 4).

Fig. 3
figure 3

Recombination analysis of strain BN23. (A) Possible breakpoints in the recombination event involving strains Thailand/S5015L/2015 and CHN-AH-2004 were identified using the RDP method and confirmed using the RDP, GENECONV, Chimaera, Maxchi, BootScan, SiScan, and 3Seq applications in the RDP program (p < 0.01). (B) Nucleotide sequence similarity was assessed using SimPlot v.3.5.1.

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Table 3 Primers and probes used for real-time PCR analysis
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Table 4 PDCoV reference strains used in this study
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CHN/CQ/BN23/2016 is highly pathogenic to newborn piglets

As PDCoV BN23 was found to be a recombinant strain that differs from the prevalent PDCoV strains, its pathogenicity was tested in piglets. Six 3-day-old piglets, free of PDCoV, PEDV, TGEV, PRV, and PCV-2, were divided randomly into two groups and inoculated with PDCoV BN23 (106 TCID50 per pig) or PBS. At one day postinfection (dpi), piglets inoculated with BN23 exhibited a loss of appetite. They also had mild diarrhea, and their body temperature decreased from about 39°C to 37°C. The piglets in the mock-infected group did not show clinical signs. At 2 dpi, the inoculated piglets developed severe diarrhea and consumed almost no milk, and their temperatures dropped to about 36 °C. All of the challenged piglets died at 3 dpi. The small intestines were found to be transparent, thin-walled, gas-distended, and filled with yellow watery content (Fig. 4A). No clinical signs were observed in the mock-infected piglets. Different tissues were also harvested for viral load determination, and the ileum was also fixed and subjected to histological analysis, which showed that the small-intestinal villi were mildly atrophied and contained aggregates of inflammatory cells. No lesions were observed in the mock-infected group (Fig. 4B). IFA analysis performed with sections showed that BN23 was present in the villus of the intestine (Fig. 4C). Real-time PCR analysis demonstrated that PDCoV BN23 has broad tissue tropism, with the viral genome detected in the duodenum, jejunum, ileum, colon, cecum, rectum, gastric mucosa, and mesenteric glands, with especially high viral loads in intestines. Within the intestines, higher viral copy numbers were found in the colon, cecum, and rectum than in the duodenum, jejunum, and ileum (Fig. 4D).

Fig. 4
figure 4

Pathogenicity evaluation in BN23-infected piglets. (A) Macroscopic lesions in BN23-challenged piglets and mock-infected piglets at 3 dpi. (B) Histologic lesions in the intestine caused by PDCoV CHN/CQ/BN23/2016. (C) IFA analysis of ileum sections. (D) Virus load in different tissues of piglets challenged with BN23. (D) Immune responses induced by PDCoV CHN/CQ/BN23/2016 infection in different segments of the intestine

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We then examined whether PDCoV infection could induce enteric immunity. The results demonstrated that the IFN-λ3 gene was significantly upregulated in PDCoV-infected small intestines. Also, PDCoV infection enhanced the expression of IL-1β, IL-12A, and GM-CSF, suggesting that BN23 could induce inflammatory reactions in the intestine (Fig. 4E).

Discussion

PDCoV is a novel porcine enteric coronavirus. The clinical signs, including watery diarrhea, vomiting, and dehydration, are similar to those caused by PEDV [31]. In 2012, two PDCoV strains were first identified in Hong Kong [5]. Since then, PDCoV has been reported in other provinces of China [13, 14, 31, 36]. A retrospective study showed that PDCoV could be detected in samples from as early as 2004. A total of 215 samples collected during 2004–2014 in Anhui, Guangxi, Hubei, and Jiangsu provinces displayed a positive rate of 6.51% for PDCoV [36]. In previous studies, the prevalence of PDCoV infection was 23.4% in samples collected from Shanxi, Guangdong, and Hubei province, and it was 33.71% (120/356) in Jiangxi provinces since 2014 [13, 31]. Those results showed that PDCoV is circulating in southern China, but few studies on PDCoV have been performed in western China. Therefore, samples from piglets with watery diarrhea in western China were collected to test for PDCoV. The results demonstrated that PDCoV strains are also highly prevalent in western China, and one PDCoV strain was later isolated.

Phylogenetic analysis was performed to examine the evolutionary history of the new PDCoV isolate CHN/CQ/BN23/2016. Its complete genome showed 97.5%-99.8% sequence identity with other Chinese strains at the nucleotide level (Supplementary Table S2). It formed an individual subcluster between the Chinese lineage and the early Chinese lineage. The early Chinese lineage also included several strains isolated in Gansu and Qinhai provinces since 2016 [37]. These two areas were also invested in this study. The frequent identification of early Chinese strains suggested that the early Chinese PDCoV strains might have circulated in western China and recombined with PDCoV strains of other lineages. The phylogenetic analysis also revealed that the 3’UTR of BN23 belongs to the United States lineage, whereas the 5’UTR belongs to the early Chinese lineage. Recombination analysis indicated that a recombination event had occurred within ORF1a of BN23. The major parent strain of BN23 was CHN-AH-2004, while the minor parent was Thailand/S5015L/2015. In western China, Sichuan province contributes greatly to the pig production industry. The five provinces investigated in this study are not the traditional pig husbandry areas. Piglets are usually transported from Sichuan to the other five provinces. Moreover, Sichuan is an important transport hub for both western China and Southeast Asia. The frequent transport of animals may result in the emergence of recombinant PDCoV strains.

An animal experiment showed that BN23 is highly pathogenic to newborn piglets. The lamina propria was heavily infiltrated by inflammatory cells such as macrophages, lymphocytes, neutrophils, and eosinophils, which is in agreement with previous studies [24, 38, 39]. However, recently isolated Chinese PDCoV strains belonging to the Chinese lineage have been found to cause much milder disease in sucking piglets. In those studies, although typical symptoms were observed in infected piglets, most recovered from the infection [40,41,42]. Our results suggest that BN23 is more pathogenic than with other PDCoV strains belonging to the Chinese lineage. Moreover, BN23 infection elevated the expression of IFN-λ3, which results in a powerful response against the infection within the intestine. Inflammatory cytokines, including IL-1β, IL-12A, and GM-CSF, were also upregulated during BN23 infection. Inflammatory reactions were also observed in previous studies and correlated with H&E straining, suggesting that PDCoV infection causes a strong immune response [43]. Although it has been reported that PDCoV infection inhibits IFN-associated response in vivo [44], the opposite phenomenon was observed in vitro. The complex microenvironment within the intestine may be responsible for this difference.

In summary, this study demonstrated that PDCoV is prevalent in western China, and a new PDCoV strain, CHN/CQ/BN23/2016, showing genetic divergence from other Chinese strains, was identified. This virus formed an individual cluster between the early Chinese lineage and the Chinese lineage and was found to be a recombinant strain. It was also found to be highly pathogenic to newborn piglets. These results provide important information on the evolution of PDCoV in western of China and suggest that more surveillance is needed.