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Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper

Sid et al. investigated the roles of RIG-I and RNF135—previously proposed to enhance immunity to avian influenza—in chickens, a species that naturally lacks both genes. By generating transgenic chickens expressing duck RIG-I and RNF135 under the control of their respective duck promoters using primordial germ cells (PGCs), the authors examined susceptibility to H7N1 infection. Reintroducing these genes increased acute inflammation and mortality following avian influenza infection. These findings suggest that the evolutionary loss of RIG-I in chickens may represent an adaptive response to viral infection.

Major comments  - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• What are the basal levels of innate immune activation (such as type I interferons) in RIG-I–proficient duck cells compared to RIG-I–deficient chicken cells? How does overexpression of RIG-I in chicken cells alter the basal inflammatory state?

• The organ-specific expression patterns of RIG-I are particularly intriguing. How does RIG-I expression in various organs of transgenic chickens compare to that in ducks? Notably, the absence of RIG-I in chicken lung cells may suggest an evolutionary adaptation to viral infections targeting the respiratory tract. This could reflect a compensatory mechanism or a selective advantage in modulating immune responses in the lungs. It would be interesting to explore and comment on this in the discussion.

• In Figure 5, RIG-I/RNF135-expressing chickens showed increasing clinical illness—from 13% of animals exhibiting symptoms at 1 dpi to 50% at 3 dpi (p < 0.05). Interestingly, this trend contrasts with the viral genome load: co-expression of both genes resulted in lower levels of viral RNA compared to chickens expressing RIG-I alone. This raises an important question: Why does reduced viral genome load not correspond with decreased clinical severity?

Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• No

Conflicts of interest of reviewers

• No

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1. Result: “Authors hypothesized a compensatory evolution of melanoma differentiation-associated gene-5 (MDA5) that accompanied the gradual loss of RIG-I in chickens ”

   • Comments: 

   • MDA5 supposedly compensates for RIG-1 loss, potentially affecting immune response when RIG-1/RNF135 is reinstated - could you discuss what results you'd expect in the absence of MDA5?

2. Result: “To date, the effect of in vivo expression of RIG-I in chickens has never been studied, and no transgenic chicken lines expressing duck RIG-I have been generated”

   • Comments: 

   • What about PMID: 39733732?

3. Result: “In contrast, co-expression of RNF135 with RIG-I seems to balance the adaptive immune cell populations,”

   • Comments: 

   • This balance is highly interesting! Could you give mor context in terms of other work investigating the interplay of RIG-I and RNF135 in other species?

4. Result: “ In the lung, challenged RIG-I-RNF135-expressing chickens displayed a virus replication rate similar to WT birds and a significantly lower replication rate than RIG-I-expressing chickens at two dpi (p<0.05). ”

   • Comments: 

   • After you have previously described the effect of RIG-I and RNF135 individually in detail, I here miss the presentation of the results of the co-expressing animals in terms of morbidity and clinical symptoms.

Competing interests

The authors declare that they have no competing interests.