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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