No evidence for highly pathogenic avian influenza virus H5N1 (clade 2.3.4.4b) in the Antarctic region during the austral summer 2022/23

The authors present work which aims to assess the presence of high pathogenicity avian influenza virus (HPAIV) in the Antarctic and sub-Antarctic regions. This work is based on results from samples taken from birds in these regions and observational mortality data. The authors did not detect HPAIV in any samples tested and report mortality is at normal levels, concluding that HPAIV is not present in these regions during the austral summer of 2022/23.

The timing of this manuscript is slightly unfortunate considering the recent detection of HPAIV H5N1 in the Antarctic region. However, this work is still of interest, if not more relevant when considering timing of introduction and describing the network and approaches which could be used for continued surveillance and monitoring in this ecologically important region. Considering that HPAIV H5N1 has now been detected in this region, the manuscript required editing of the introduction and discussion to reflect this event. This reviewer purposes the following changes.

Major changes:
1. The manuscript, mainly the introduction and discussion require changing in light of the detection of HPAIV in the Antarctic region https://doi.org/10.1101/2023.11.23.568045. This pre-print needs acknowledging in the context of this work.
2. Regarding the testing conducted, and considering all the different approaches used, are the authors confident in the ability to detect HPAIV?
3. It is very unclear which methods have been used for detections, only a few reference are provided for individual assays, the text provided is excessive and often includes doi number in some cases without refences, such as on page 14 line 37 for testing at Laboratorio de Virologia Molecular. In addition, no details for testing at all are provided in page 11 line 25 for testing at Animal and Plant Health Agency, UK.
4. The title is ‘no evidence of HPAIV virus H5N1 (clade 2.3.4.4b)’. From the sampling conducted and assays used it is not clear if these assays are specific for this clade or HPAIV in general as this is used interchangeably in the manuscript. If more broad assays are used, a more accurate title would be no evidence of HPAIV.
5. For the observational data, it is not clear how are the authors attributing increased mortality over baseline. also is this targeting species of highest risk? More discussion needed in this area.

Minor changes
Title
6. Line 4; 2022/23 could be somewhat misleading to also imply the summer of 2023. Especially since the recent detection of HPAIV was in the summer of 2023. Please change to be more precise.
7. Use high pathogenicity avian influenza not highly pathogenic avian influenza virus as per WOAH standards.
Affiliations
8. There are two institutions listed for ‘5’ in the affiliations and no ‘6’
Abstract
9. Line 58; surveillance activities are not limited to ‘H5N1’ HPAIV, but rather HPAIV in general, please change.
10. Line 59; HPAIV testing not HPAI testing.
11. Line 59; testing was not on islands only, some samples in Figure 2 are from continental Antarctica, please amend.
12. Line 60; please define the timeframe of the study
Main text
13. Line 63; define ‘profound’, reword and use metrics to support this statement.
14. Line 64/65; imprecise language with ‘most continents’.
15. Line 66; is refence 3 the most relevant for increase replication in wild birds? A better refence to support improve replication in wild birds would in vivo experimental studies in wild birds such as https://doi.org/10.1099/jgv.0.001852 or https://doi.org/10.1080/03079457.2023.2196258.
16. Line 68; reference 3 isn’t appropriate.
17. Line 83-86; refence needed to support this.
18. Line 90; who are 'experts', this needs more explanation.
19. Line 94; how are the authors defining the Antarctic and sub-Antarctic region? This pre-print defines as within the Antarctic Convergence https://doi.org/10.1101/2023.11.23.568045. The authors need to define this.
20. Line 106; have the authors assayed for influenza virus, from the appendix only PCR appears to have been used, which is a measure of viral RNA presence only. The authors need to confirm. Also line 109.
21. Have the authors prioritised testing and observational data based on which bird species are of most likely to introduce HPAIV into these regions. The authors include reference 14, which discusses highest risk species, but have not included this in the introduction.
22. Line 125; refence 3 is not needed here.
23. Line 145; needs refences, suggest reference 14.
Technical appendix
1. Observations section: needs formatting with bullet-points addressing, text is across multiple lines.
2. Observations section: Line 36: new symptom associated with current outbreak. Animals do not have symptoms, they have clinical signs. Also refence needed to support this. I presume this is the refence in question https://doi.org/10.1111/ibi.13275.
3. Field and laboratory protocols: Which methods have been used for detections, few reference are provided for individual assays, the text provided is excessive and includes doi number in some cases with no refence, such as on page 14 line 37 for testing at Laboratorio de Virologia Molecular.
5. Line 25, AB, I presume Ashley Bennison, is listed as performing qPCR at Animal and Plant Health Agency, UK, but this is not listed in Ashley Bennison’s affiliations.