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Peer Review of : Pathology of Influenza A (H5N1) Infection in Pinnipeds Reveals Novel Tissue Tropism and Vertical Transmission 

Reviewer: Leen Arnaout

Co-reviewer: Mam Roheya Jack

This manuscript outlines the pathology of highly pathogenic avian influenza (HPAI) H5N1 infection in pinnipeds along the Argentinean coast. The authors present clinical, pathological, and molecular findings from necropsies conducted on three South American sea lions (Otaria flavescens) and one Southern elephant seal (Mirounga leonina). Using such observations, the authors claim to unravel novel systemic impacts and novel tissue tropism of HPAI H5N1 in pinnipeds, specifically in nervous system tissue. The authors also provide the first evidence of vertical transmission of this virus in pinnipeds.

I believe this paper is a crucial initial contribution to the growing field of HPAI H5N1 pathology in sea mammals, possibly being a foundation for future works investigating specific mechanisms of H5N1 pathogenesis and organ damage. The authors clearly identified a knowledge gap in HPAI H5N1 pathobiology in pinnipeds, and outlined a solid diagnostic approach to identify novel pathological signs through sample visualization, and immunohistochemistry and RT-sqPCR to confirm viral infiltration of the tissue. 

However, there are various major concerns with this manuscript:

• Contradiction between the presented findings on H5N1 damage to the lungs of the studied necropsies, and the discussion of such results: based on reported antibody staining results, HPAI-related lesions in lung tissue were identified in only one out of four seals (lines 235-236, 239-240), and AIV-positive staining of epithelial cells lining the embryonic airways of the fetus (lines 227-229). Yet, in the discussion section, the authors claim that H5N1 causes widespread lesions and infection in the lungs (lines 290-294). Therefore, I recommend the authors remove the language around H5N1’s causality of the respiratory pathological findings, and instead note it as a possible contributor to those findings that should be corroborated with further molecular analyses.

• Assumptions of causality, and limited discussion of confounding factors: inflammation and damage in the lungs, and myocarditis, could have been caused by stress or secondary bacterial infections rather than H5N1. A more detailed discussion of those possible confounding factors, and the differential behind determining H5N1 causing these pathological findings, is crucial. For example, to definitely rule out other possible causes for the lung lesions, the authors could conduct further experiments like microbial culturing of the lesions to rule out bacterial infections. This could be expanded to rule out causes for other pathological findings, too.

• Small sample size: four necropsies are not enough to make generalizable statements on the physiological impacts of H5N1 infection in pinnipeds, especially in the case of contradicting findings regarding infiltration (or lack thereof) of the virus in the lungs. However, this is understandable due to the limited number of pinnipeds corpses found, especially those whose bodies were still “fresh” and able to be studied. Therefore, I recommend the authors revise the language around the generalizability of H5N1 tissue tropism patterns in pinnipeds, based on this sample size. For example, instead of stating very generalizable claims about H5N1 causing myocarditis and damage to the nervous system in pinnipeds, the authors could state that they found evidence supporting these pathological findings based on the necropsies conducted, and that further necropsies are required to substantiate these claims of H5N1 impact on pinniped physiology in general.

• Sampling methodology: when full necropsies were unable to be carried out, samples of the CNS and lungs were collected through the foramen magnum and thoracic windows respectively, which does not allow for sampling tissues from the different parts of these respective organs. This was the case for 2 out of 4 pinnipeds in this study, and thus decreases the reliability of the claim that H5N1 infection infiltrates the lungs and CNS–especially in the case of the lung samples, where anti-HA antibodies were absent in almost all lung samples. While the authors addressed this in their publication, the authors should attempt full necropsies for future publications.

In addition, minor discrepancies in the years of publication of some papers were found. For example, Spackman et. al. was reported as published in 2022, when it was done in 2002.

Overall, I believe this article is an important contribution to the growing field of HPAI H5N1 pathology in sea mammals, presenting some preliminary evidence of novel impacts of the virus on various organ systems, and serving as a foundation for future research on more granular mechanisms of H5N1.

Competing interests

The authors declare that they have no competing interests.