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 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
have a Q642K, like those in Vietnamese and Thai strains. These results
provide evidence 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 isolate 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 provided evidence 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 isolate 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 isolate emerged from a 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 have been used
to estimate the genetic relationship of PDCoV strains worldwide.
Perez-Rivera et al. demonstrated that Mexican PDCoV formed two clades:
the group including strains isolated in 2015, and those 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, the 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 isolate revealing that this isolate has undergone phenotypical
changes after its emergence in North America. Although these
substitutions were not 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 import of a large number of purebred
animals (~ 150 tons) during the 2014 and 2018 period.
Furthermore, Peru imports feed ingredients for swine farms mainly from
the US (MINAGRI, 2020). Altogether, this might explain the possible
routes for PDCoV entrance into the country, similar to that described
for other viruses of importance for swine industry (Dee et al., 2018;
Ramírez et al., 2019). Interestingly, there is no report of PDCoV in
other South American countries so the introduction from those 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 isolate
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 Drs. Mariluz
Arainga-Ramirez, and Steven Lawson for their 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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