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 positive to Porcine Deltacoronavirus
with a cycle threshold (Ct) value between 9-14, revealing a high viral
load, while testing negative 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. A fluorescence test using an anti-N
monoclonal antibody confirmed viral replication. 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 large group of single-stranded, positive-sense
RNA viruses that infect a broad range of species such as avian and
mammals, including humans. Coronaviruses belong to the orderNidovirales , family Coronaviridae , subfamilyCoronavirinae . 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 and Deltacoronaviruses 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 signs from other forms of enteric disease
such as PED, TGE and Swine Acute Diarrhea Syndrome (SADS). 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 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 has
never been reported in South America. Hence, we report the first
isolation and whole genome sequencing of PDCoV in Peru, providing new
insights into the molecular epidemiology of this emerging disease in
swine.
Materials and methods