3.5. Peruvian PDCoV Spike amino acid sequence reveals unique
substitutions compared to other PDCoV strains
As we observed multiple changes at the nucleotide level in the S gene,
we were interested in evaluating whether these changes represent
modifications at the protein level. Thus, we found multiple changes in
the S protein sequence (see Figure 4B). Most relevant changes are found
K96R, G552E, A630V and V1052A that represent unique variations compared
to other PDCoV strains. Other amino acid changes have been also found in
Chinese strains such as P38L and A137V. We also detected F530L, like the
one found in the Vietnamese strains. Furthermore, the Peruvian strains
has a Q642K, like those in Vietnamese and Thai strains. These results
evidenced that PDCoV has undergone to unique changes that indicate a
degree of genetic diversity in the Peruvian PDCoV strain. Based on
previous studies of PDCoV Spike protein characterization (Shang et al.,
2018), amino acid substitutions observed here are located randomly
across both S protein subunits (S1 and S2) but none of them is located
in the RBD region (S1-CTD).
Discussion
PDCoV is one of the most recent and relevant coronaviruses of swine
industry. It represents a major threat for swine productivity, and it is
responsible for large economic losses worldwide. Yet, PDCoV remains
poorly studied despite major efforts made recently. In Peru, multiple
cases of enteric disease have occurred, however, these cases are not
properly addressed and frequently misdiagnosed. Hence, this study
represents the first report, isolation and phylogenetic characterization
of a Peruvian PDCoV strain using the whole genome sequence and its major
S protein, revealing unique aspects compared to other PDCoV strains.
Our viral isolation findings contrasted with those detected by qRT-PCR.
This evidenced that a large proportion of viral particles were unable to
replicate into a cell line support. This has also been reported by
others indicating that low viral isolation rates might be attributed to
sample degradation and viral viability (Hu et al., 2015). On the other
hand, multiple authors have shown that successful viral isolation are
due to other factors such as cell line permissibility and enzyme
treatment (Yang et al., 2020; Zhao et al., 2019). In our study, it
remains unclear whether trypsin concentration (10 µg/ml) might have
played a role in the lack of viral replication in Vero cells. Additional
studies, at different trypsin concentration, will elucidate Vero cell
line permissibility to PDCoV. Interestingly, we did not observe cell
toxicity in our assays, as that was absent following the first 5 days of
inoculation and PDCoV cytopathic effect was evidenced in the second
passage. These results contrast with other claims that cell toxicity is
common during PDCoV isolation using intestinal content or fecal samples
(Hu et al., 2015). Nevertheless, further studies are required to clarify
the implications of cell permeability to PDCoV in viral replication and
its effects on clinical presentation.
Whole genome analysis revealed that our strain was closely related to
North American strains. The close relationship within the Peruvian and
the North American strains indicate they share a common phylogenetic
ancestor and revealed that the Peruvian strain emerged from an US
strain. In 2016, Perez-Rivera et al. reported the first phylogenetic
analysis of PDCoV in Mexico, focusing the analysis on the S gene
nucleotide sequence, and no report of PDCoV whole genome sequence was
made (Pérez-Rivera et al., 2019). Thus, we were unable to evaluate the
phylogenetic relationship using the whole genome sequence of PDCoV from
Peru and Mexico. We believe that this would add deeper understanding
about its appearance in Peru and contribute to its epidemiology in South
America.
Due to its high variability, S gene nucleotide sequences has been used
to estimate the genetic relationship of PDCoV strains worldwide.
Perez-Rivera et al. demonstrated that Mexican PDCoV formed two clades:
the group with the strain isolated in 2015 and the PDCoV isolated in
2017 (Pérez-Rivera et al., 2019). In our study, phylogenetic analysis
using S gene nucleotide sequences reveals that Peruvian PDCoV grouped
closely to the Mexican strain isolated in 2015 within the North American
phylogroup. This indicates a close relationship among PDCoV strains from
Mexico, US and Peru, sharing a common ancestor and evidencing a
dissemination route of PDCOV from North America to South America.
Interestingly, multiple non silent mutations were found in the Peruvian
PDCoV strain compared to other genomes. Even though some these mutations
have been described in other PDCoV strains, some are unique to the
Peruvian strain revealing that this strain have undergone phenotypical
changes after its emergence in North America. Although these
substitutions were no located in critical regions of glycosylation sites
nor in the RBD region (S1-CTD), they might have influence in
ligand/receptor interaction. Further studies are required to clarify
whether these modifications have implications in the pathobiology and
development of the clinical disease.
To date, it is unclear how PDCoV was introduced in Peru. However, there
is a long history of commerce between Peru and North American countries
that has expanded in recent years. The National Service of Animal Health
in Peru (SENASA) reported the importation of a large number of purebred
animals (~ 150 tons) during the 2014 and 2018 period.
Thus, this might suggest that PDCoV introduction was through semen, food
and purebred animals trade or through people acting as fomites, from
places with PDCoV like that described for other viruses of importance
for swine industry (Dee et al., 2019; Ramírez et al., 2019).
Interestingly, there is no report of PDCoV in other South American
countries so the introduction from neighboring countries is unlikely.
Nevertheless, further studies are needed to understand the epidemiology
of this disease in Peru and its relationship to other countries.
In conclusion, Peruvian PDCoV strain was successfully sequenced,
isolated and phylogenetically analysed demonstrating that this strain
has been derived from a US strain. To our knowledge, this is the first
report of a PDCoV strain detected in South America and offers new
insights about the epidemiology of PDCoV worldwide.
Acknowledgement
The authors would like to express their gratitude to Dr. Mariluz Arainga
for her contribution, and the Universidad Nacional Mayor de San Marcos
for its financial support. We also would like to acknowledge the farm
professionals and the national health authorities (SENASA-Peru) for the
information provided.
Conflict of interest
All authors have declared no conflict of interest
Ethical statement
The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to. No
ethical approval was required as research samples were obtained in
accordance with guidelines from the Peruvian National authorities in
animal health.
Data availability
The data that support the findings of this study were submitted to the
GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) with accession
number MT227371 for the Peruvian strain of PDCoV obtained.
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