Review 2: "Validation of Optimised Methods for Avian Influenza Virus Isolation in Specific Pathogen-free Embryonated Fowls’ Eggs"

RR\ID Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

***************************************

Review: The study's concept of finding a method to rapidly evaluate avian influenza presence and providing a good biosecurity bill of health for farm is one that I support in business continuity and promoting and protecting livelihoods. The authors claim that a reduction of 2 days wait time can be achieved without losing effectiveness and safety.

My main concern with this study is with the selection of negative and positive controls. A spectrum of HPAIVs with slow, medium and fast-growing rates would be more convincing. Selection of a single HPAIV may not be representative enough. The reference list is also inadequate as more citations are needed to support the statements within the manuscript.

Below are some line edits:

• Figure 1:

  • “approaches (‘1+1’, ‘1+2’ and ‘1+3’)”: This is missing '2+2'

  • How does one certify eggs are negative prior to inoculation? Should negative controls be virus free eggs from another country if a national outbreak is ongoing?

• Materials and Methods:

  • “…inoculated undiluted into EFEs using each of the 117 shortened models as described in Table 1.”: The challenge with using 1 strain as a positive control is that it may NOT be representative of a slower growing strain. Faster growing AIV strains tend to have a higher titer in tissues, giving a higher inoculum/viral load. What type of strain was H5N8 a slow or fast growing strain?

  • “For each NAD investigation, the statutory AI testing algorithm 159

    with VI, reverse transcription polymerase chain reaction (rRT-PCR) and serology 160 described previously was employed (Reid et al., 2019)”: What if another virus was introduced and circulated without tropism?

• Table 1:

  • There may be an experimental design issue as this is representative of a single strain not a specturm of AIVs.