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