PREreview of Genetic and Glycosylation Variability of the GP5 Glycoprotein in NADC34 Variant Strains of Betaarterivirus americense from Lima, Peru

General Assessment

The study provides valuable molecular epidemiological data on the NADC34 variant of PRRSV-2 in Peru, focusing on the GP5 protein—a key target for neutralizing antibodies. The identification of a potentially new N-glycosylation site (N57) and the detailed mapping of mutations within known epitopes are the primary strengths of the work. The manuscript is generally well-structured, but it could be strengthened by clarifying certain statistical interpretations and improving the visual presentation of the data.

Major Comments

1. Statistical Interpretation (Tajima’s D)

• Observation: In lines 201–204, the authors report a negative Tajima’s D value (-0.78746) and suggest this indicates "high genetic variability" or "population subdivision."

• Recommendation: Technically, a negative Tajima’s D typically indicates a recent population expansion or purifying selection following a bottleneck, leading to an excess of rare alleles. While it does reflect diversity, the authors should be more precise in their evolutionary interpretation. Is the negative value significant? The text says "no significant deviation (P > 0.10)." If the value is not statistically significant, the authors should be cautious about using it to draw strong conclusions about population dynamics.

2. Functional Implications vs. Speculation

• Observation: The manuscript frequently links the observed mutations and glycosylation patterns to "immune evasion" and "vaccine failure" (e.g., lines 238, 265, 272).

• Recommendation: While these links are well-supported by general PRRSV literature, they remain speculative for these specific 24 strains since no in vitro neutralization assays were performed. The authors should explicitly state in the "Limitations" section (or Conclusion) that functional assays are required to confirm if these specific NADC34 variants indeed escape current commercial vaccines used in Peru.

3. The "Unreported" N57 Site

• Observation: The authors highlight N57 as a "previously unreported site" (line 194).

• Recommendation: This is a significant finding. The authors should expand on this in the Discussion. Does this site appear in any international databases (GenBank) for NADC34 strains outside of Peru? A quick comparison with recent NADC34 sequences from China or the US would help determine if this is a unique "Peruvian signature" or a broader emerging trend in sublineage 1.5.

4. Table 1 Presentation

• Observation: Table 1 (page 15) lists the N-glycosylation sites.

• Recommendation: It would be much more informative to add a column for "Total Number of Sites" per strain. This would allow the reader to quickly see that strains 40 and 41 are "hyper-glycosylated" (6 sites) compared to strains 36–39 (2 sites), which is a point emphasized in the text.

Minor Comments

1. Figure 3 (Phylogenetic Tree)

• Observation: The circular tree is visually appealing but the labels in the "turquoise" branch are very small and difficult to read in the current format.

• Recommendation: Consider providing a "zoomed-in" rectangular version of the Lineage 1.5 cluster as a panel B, or increase the font size of the Peruvian study strains to make them stand out from the reference sequences.

2. Figure 4 (Alignment)

• Observation: The alignment is comprehensive but spans two pages and is quite dense.

• Recommendation: Highlight the "N57" site specifically in the figure or with an arrow, as it is one of the key findings. Also, ensure the color-coded legend for epitopes (A, B, C, etc.) is clearly defined in the figure caption to avoid the reader having to hunt for it in the text.

3. Terminology and Consistency

• Line 36: You mention a negative Tajima’s D. Ensure the minus sign is a proper mathematical symbol (–) rather than a hyphen (-) for consistency with scientific formatting.

• Lines 153-154: You mention "nine isolates previously reported... in 2019 and 2025." Given the preprint date says 2026, please ensure the timeline of "previous reports" is clear to the reader (especially regarding the 2025 citation).

4. Materials and Methods

• Line 103: Mentioning "Chromas Lite®" is good for transparency. Did the authors also use a specific tool for the multiple sequence alignment (MSA) before the tree construction? (e.g., ClustalW, MUSCLE, or MAFFT). This should be explicitly stated.

Conclusion

This is a solid molecular characterization paper. By addressing the statistical nuances of the evolutionary analysis and clearly distinguishing between observed genetic traits and predicted functional outcomes, the authors will improve the impact and accuracy of the manuscript.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.