Review 1: "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.

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Review: The manuscript reports the evaluation of AIV isolation studies in SPF embryonated fowls’ eggs in testing 1 and 2 egg passages with 1-2 d pi in the 1^st egg passage and 1-4 d pi in the 2^nd egg passage. The data generated from this study is relevant and important in the field of AIV diagnostics.

However, the study experiments were not well designed, such as, all test samples were old samples collected during AI outbreaks many years ago, which would affect test results due to longtime storage with unknown storage conditions (-80C or -20C?) and changes of virus survival status. Thus, those samples were not sufficient for a validation test. Refer to validation test requirements to make appropriate improvements accordingly to meet the study purpose.  

The manuscript was not written well scientifically, major improvements should be made in the following areas as:

1. Figure 1 was not appropriately designed and should be removed. VI in two egg passages can be briefly described to be easily understood without the Fig. Note: the incubation time of VI in each egg passage cannot be less than 48 hours, days (better in hours) of egg incubation time are counted by each passage separately but not continuing count from 1^st to 2^nd passage.

2. The test procedures for two egg passage models were not clearly described, such as line 125-127 “The HA test was performed on one day 1 egg from day 0 (P1) and 200µl supernatant fluid was inoculated back into P2 (3 x eggs for the ‘1+1’, ‘1+2’ and ‘1+3’ models using a total of 9 eggs)”. It’s well-known that AIV isolation requires 1 to   2 egg passages, so just describe what’s your procedures for the 1^st and 2^ndegg passage, not to make complicated   ‘1+1’, ‘1+2’ and ‘1+3’ models.

3. Table 1, need to redesign to show your experiments for 1^st egg passage and 2^nd egg passage procures, provide the number of eggs and days pi of each passage. Note: one egg’s CAF is not sufficient to pass for the next egg passage due to possible basis of a single egg, minimum 2-3 eggs’ CAF pool are required to avoid a single egg’s basis.

4. Table 2, 3, and 4, need to redesign to simply show the data in Table contents, not to add too many notes to hardly read and understand. Most of the table notes should be the table contents to be read/viewed directly.

5. Keywords: “avian influenza virus isolation in embryonated fowls’ eggs” and “notifiable avian disease virus isolation in embryonated fowls’ eggs”, which are not appropriate for key words, remove them and keep “notifiable avian disease; embryonated fowls’ eggs”. Note: the term “fowls’ eggs” is not appropriate, too, fowls refer to all avian species but not a specific avian species, if chicken eggs were used in this study, it should be changed to “embryonated chicken eggs”.