Abstract
Porcine Deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America. Yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru; was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positives to Porcine Deltacoronavirus with a cycle threshold (Ct) between 9-14, revealing a high viral load, while tested negatives to Porcine Epidemic diarrhea and Transmissible Gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. One sample was processed for whole genome sequencing (NGS). In short, raw reads were imported into CLC genomics and assembled de novo . Out of 479k reads generated from the sample, 436k assembled into a 25501 bp contig which was 99.5% identical to a reference Porcine Deltacoronavirus strain from US within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared to previously described Porcine Deltacoronavirus strains. Altogether, our findings represent the first report of Porcine Deltacoronavirus in South America, its genomic characterization, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of Porcine Deltacoronavirus infections in the swine industry.
Keywords: Porcine Deltacoronavirus, Peru, diarrhea, whole genome sequencing, PDCoV isolation. Emerging diseases, Veterinary epidemiology
Introduction
Coronaviruses comprise a group of single-stranded RNA, positive sense viruses that infect a broad range of species such as avian and mammals, including humans. Coronaviruses belong to the order Nidovirales , family Coronaviridae , subfamily Coronavirinae . These enveloped viruses are the largest RNA viruses identified to date ranging from 24-32 kb. Members of the subfamily coronavirinae have been recently grouped into four genus as Alphacoronaviruses ,Betacoronaviruses , Gammacoronaviruses andDeltacoronaviruses by the International Committee for Taxonomy of Viruses (ICTV) (Lefkowitz et al., 2018; Woo et al., 2010). Interestingly, it appears that the first two groups have originated from bats, while the latter two emerged from wild birds (Woo et al., 2012).
Porcine deltacoronavirus (PDCoV) is an emergent virus that causes gastrointestinal disease such as diarrhea, vomiting, dehydration and death in young piglets representing a major threat to swine industry (Jung et al., 2015; Li et al., 2019; Zhang, 2016; Zhao et al., 2019). Although PDCoV by itself causes enteric disease, co-infections with other coronaviruses such as Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) or other viruses are commonly found (Dara et al., 2018; Feng et al., 2017; Marthaler et al., 2014; Niederwerder, 2018; Song et al., 2015). In this context, PDCoV shows indistinguishable clinical sings from other forms of enteric disease such as PEDV or TGEV. Thus, proper differential diagnostic relies on genetic detection-based assays that offers a highly sensitive and specific method.
PDCoV has a unique genomic organization. Starting from 5’-end, PDCoV has a 5’ untranslated region (UTR), replicase (ORF 1a/b), spike (S), envelope (E), membrane (M), non-structural 6 (NS6), nucleocapsid (N), NS7 genes and 3’-UTR (Woo et al., 2010; Zhang, 2016). Interestingly, PDCoV lacks ORF3 and NS1, present in other well-known coronaviruses (Si et al., 2020). From these genes, S gene encodes a highly glycosylated protein responsible for binding, cell attachment and entry into the target cells and therefore highly immunogenic. Thus, S gene is commonly used for phylogenetic analysis and vaccine development.
The first identification of PDCoV dates back to 2012 in Hong Kong by Woo et al, followed by multiple outbreaks in the US (Homwong et al., 2016; Marthaler et al., 2014; Wang et al., 2014). Later, PDCoV was reported in Canada (Niederwerder, 2018), Korea (Jang et al., 2018; Lee et al., 2016; Lee & Lee, 2014), Japan (Suzuki et al., 2018), Thailand (Lorsirigool & Adthakorn, 2017; Saeng-Chuto et al., 2016) and Vietnam (Le et al., 2018; Saeng et al., 2019). More recently, PDCoV was detected in Mexico (Pérez-Rivera, et al., 2019). In South America, coronavirus infections in porcine are commonly reported. For instance, multiple studies revealed that PEDV and TGEV are present in several countries such as Colombia (Piñeros & Mogollón Galvis, 2015), Ecuador (Barrera et al., 2017) and Peru (Castro-Sanguinetti et al., 2017). However, PDCoV detection has never been reported in South America. Hence, we report the first isolation and whole genome sequencing of PDCoV strain from Peru which provides new insights into the molecular epidemiology of this emerging disease in swine.
Materials and methods