Referee 2's Review:

The paper entitled ‘The efficacy of tetracyclines in peripheral and intracerebral prion infection’ by De Luigi et al. presents novel data on the efficacy of tetracyclines in vivo in an experimental model of prion disease in hamsters.
The manuscript is well written and presented. The subject is also illustrated well enough to be readable by non-specialists. The authors have carried out rigorous experiments using the best practices for in vivo studies.

The authors’ knowledge of and background in the field of prion disease place this research among the most recent developments in pharmacological treatment of prion disease and the results are described considering the most current literature in the field. Literature cited is very comprehensive and describes what is currently known of drug discovery in prion research.

The manuscript describes interesting results of various efficacious routes of infection and treatment aimed at interfering with prion disease in vivo. While the most pronounced effects are shown when treatment is carried out close to time of infection, results presented on prolongation of incubation times once treatment is carried out at symptomatic stage of the disease are encouraging. These data are preliminary and further investigation should be carried out, in particular with respect to dosage. Why have the authors utilized 10 mg/Kg/day? If this dose is the result of previous investigation, this fact should be clearly addressed in the manuscript.

This paper should be of immediate interest to any scientist involved in prion research. Prion diseases are still incurable maladies and tremendous efforts have been made in recent years in finding effective pharmacological intervention. Moreover, its impact may be of interest to a broader scientific audience since other neurodegenerative diseases may benefit from successful and proven pharmacological strategies in the prion field.

The authors should also consider using mouse models of prion disease. The broad efficacy of tetracycline treatment should be tested and challenged with different animal models and prion strains. It is well documented in literature that different prion strains may possess dissimilar propagation properties.

The data obtained support the conclusion of the work. Nevertheless, as many other models of prion research, it heavily relies on the characteristic protease resistance of prions. What about strains that do not have PK resistant PrPSc? The authors should discuss this issue in a revised version of the manuscript.

It is mentioned in the discussion that the mechanism by which tetracyclines may act in order to prolong incubation times in vivo is not known. In fact, the authors correctly address the known antimicrobial activity of this class of drugs. It would be important to suggest a way to discriminate such activity.

An experiment with animal models maintained in a SPF facility might provide direct evidence of the efficacy of these drugs in halting prion replication in vivo.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.