Evaluation of Baloxavir Marboxil and Peramivir for the Treatment of High Pathogenicity Avian Influenza in Chickens

Round 1

Reviewer 1 Report

The authors tested two antivirals that have been used in humas for the treatment of influenza inflections. These antivirals were tested in chickens as an initial avian model to determine protective efficacy, with the ultimate goal of using the drugs in other avian species, specially those that are endangered or of high value.  The authors designed a series of experiments that lead them to the conclusion that baloxavir marboxil could potentially be the most effective drug when administered right after infection.

Comments and suggestions

• Sentence from lines 45 – 48 does not read very well.
• Line 47: delete “; therefore, in many countries”
• Line 48: add “in many countries “ after poultry so the sentence reads better.
• Line 60: please replace “this study” with “these studies”
• Line 61: Would the authors been referring to lack of mortality? This is odd since HPAIV causes embryo mortality.
• Lines 74 - 76: Please specify the dosage administered per bird and the volumes used for each drug.
• Line 78: please delete “An”. Also add a “,” after “strain”.
• Line 79: add a “,” after “Japan [21]”.
• Line 80: use “embryonated chicken eggs” instead.
• Line 95: replace the “;” after “PR” with a period to start a new sentence.
• Lines 97-98: delete “of the inoculum”.
• Line 110: please replace “inoculum” with “challenge virus”.
• Line 112: please add “out” between “four” and “of”.
• Line 114: delete “For experiment 3” since this paragraph is included in such section.
• Line 179: figure 1b shows an * for the comparison between the PR and the control groups. According to the legend *=p<0.05 which is significant, but the text in this line says there is no difference between groups. Please modify either the sentence in line 179 of the figure accordingly to accurately reflect your findings.
• Line 180: delete “completely”.
• Figure 1a: the Y axis legend should read “Number of chickens” instead.
• Line 196: it should read “a HPAI” instead of “an HPAI”, please check the test for similar mistakes and correct them all.
• Line 199: Perhaps the authors can replace “who” with “which all”, and dele “all” that comes after “had”.
• Lines 201-202: delete “of chickens”.
• Figure 2b: How were the statistics done here? Were the tissue titers from each group compared to the tissue titers across groups? Is the figure reflecting only a summary of the analysis results? If that was the case, please indicate so in figure legend. Please indicate so for each figure that involves statistical analysis.
• Line 213: please replace “administration” with “administrations”
• Line 214: delete “the” at the beginning of the line and replace “of” with “with” before “HPAIV”.
• Line 220: move “treated” between “four” and “chickens”. Also, add “most of the” after “Viral replication in”.
• Line 221. Please replace “low-titer virus was” with “low virus titers were”.
• Figure 3a: Perhaps the authors would like to select a different color scheme as all the treatment groups have similar colors.
• Line 228: please add “of” after “Effects”.
• Line 264: please add a period after mg/kg to start a new sentence. Also, add “ranging” after “titers”.
• Line 265: you could add “the” after “on”.
• Line 269: please replace “symptoms” with “signs”.
• Line 289: replace “vaccine” with “vaccines”.
• Line 290: add a period after the references on that line and start a new sentence.
• Line 292-293: perhaps the sentence should read “Additionally, vaccines against AI are usually subtype- or strain-specific, whereas AI outbreaks are not predictable for such a vaccine specificity…”
• Line 295: replace “this” with “the”.
• Line 296: delete “the” before “chickens”. Also, “the most susceptible avian species to AIV” may not be completely true. Quails are even more susceptible than chickens. I would use "a susceptible species" instead.
• Line 298: I would use “Our model demonstrates…” instead of “This demonstrates”.
• Line 304: add a period after chickens to start a new sentence.
• Line 306: I would use “effects induced by BXM” instead of “effects for BXM”.
• Line 323: replace “in” with “from”.
• Lines 340-342: Please rephrase the sentence “Using the chicken and duck models, the administration of 2.5 mg/kg of BXM induced sufficient plasma concentration of BXA to protect birds from HPAI infection for 48 hours.”. The way it is written, it misleads to the conclusion that the birds were only protected for 48 hrs instead of referring to the period of time in which the drug's plasma levels were maintained.

Author Response

Revision report

Dear Editor and Reviewers,

We appreciate your great contribution to improving the quality of the manuscript we submitted in Viruses for publication. In the previous version, the manuscript was titled “Evaluation of baloxavir marboxil and peramivir for the treatment of highly pathogenic avian influenza in chickens” (ID Viruses-1015663) and it has been revised. In the current version, the title was slightly modified as “Evaluation of baloxavir marboxil and peramivir for the treatment of high pathogenicity avian influenza in chickens”, this has been changed according to the recent recommendation by the World Organisation for Animal Health (OIE).

All modifications according to the reviewers’ comments are made as tracking mode as per journal recommendation. Two new references have been inserted in line 62 and 343. In lines 61-64, the references have been permutated according to each statement.  Minor modifications were made in the Figures according to the reviewer’s comments.

Reviewer #1

• Comment 1. Sentence from lines 45 – 48 does not read very well
• Comment 2. Line 47: delete “; therefore, in many countries”
• Comment 3. Line 48: add “in many countries “ after poultry so the sentence reads better.

Answer: Thank you for the suggestions, the paragraph has been rewrite combining the comment 1,2 and 3 in lines 45 to 48 as follows “Given that the vaccine must be frequently updated due to the antigenic changes of viruses occurring in each epidemics [11,12], also the vaccine may lead to the silent spread of HPAIV with asymptomatic infections [13]; vaccination against HPAI is prohibited for use in poultry in many countries [8,14].

• Comment 4. Line 60: please replace “this study” with “these studies”

Answer: The word was replaced as follows “in these studies: a lack of clinical signs in chickens when challenged with a low pathogenic avian influenza virus” 

• Comment 5. Line 61: Would the authors been referring to lack of mortality? This is odd since HPAIV causes embryo mortality

Answer: Thank you for this important observation. After checking carefully, we have modified the statement as follows” presence of virus growth and embryo mortality but not clinical signs as it can be observed in adult birds, when eggs were used for HPAIV infection”.   

• Comment 6. Lines 74 - 76: Please specify the dosage administered per bird and the volumes used for each drug.

Answer: The detail for dosage and volumes were provided as follows “For drug administration to birds, a tablet of BXM was crushed into powder and was further dissolved in 2 mL of sterile phosphate-buffered saline (PBS) and the volume was calculated accordingly (e.g. 1 ml for 10 mg, 100 µL for 1 mg, 50 µL for 0.5 mg and so on). For PR, 1 mL containing 10 mg was administered.”

• Comment 7. Line 78: please delete “An”. Also add a “,” after “strain”.

Answer: The “an” was deleted at the beginning of the sentence “HPAIV A/black swan/Akita/1/2016 (H5N6) (A/BS/Akita/1/16) viral” 

• Comment 8. Line 79: add a “,” after “Japan [21]”.

Answer: The coma was added in the sentence after Japan as suggested.

• Comment 9. Line 80: use “embryonated chicken eggs” instead.

Answer: The position of words was changed as follows “embryonated chicken eggs”

• Comment 10. Line 95: replace the “;” after “PR” with a period to start a new sentence

Answer: The sentence was modified by adding the period “A dose of 20 mg/kg was immediately administered orally for BXM and intramuscularly for PR. The treatment was repeated every 12 hours for 5 days”.

• Comment 11. Lines 97-98: delete “of the inoculum”.

Answer: The word inoculum was deleted in the sentence as follows” … however, because of the high dose (10 CLD50) of the challenge virus, the doses were adjusted accordingly.

• Comment 12. Line 110: please replace “inoculum” with “challenge virus”.

Answer: Thank you for the suggestion, the word inoculum was replaced with challenge virus as shown “Chickens were infected with 100 µL of the challenge virus, and a single dose of BXM was orally administered immediately after the infection”

• Comment 13. Line 112: please add “out” between “four” and “of”.

Answer: The word “out” has been added as follows “In all three experiments, organ samples including the trachea, lung, kidney, colon, and brain were collected at 3 dpi from four out of eight chickens in each group for virus recovery”

• Comment 14. Line 114: delete “For experiment 3” since this paragraph is included in such section

Answer: The sentence was revised as follows “In addition to sample collection from organs at 3 dpi…”.

• Comment 15. Line 179: figure 1b shows an * for the comparison between the PR and the control groups. According to the legend *=p<0.05 which is significant, but the text in this line says there is no difference between groups. Please modify either the sentence in line 179 of the figure accordingly to accurately reflect your findings.

Answer: Thank you for this important observation, the statement been modified following the result of this experiment as follows “however, in PR group, viral replication was noticeably higher even though the titers did not reach those observed in the control group”

• Comment 16. Line 180: delete “completely”.

Answer: The word completely was deleted as follows “As per our findings, BXM treatment was able to suppress viral replication, enabling the chickens to survive infection, …”

• Comment 17. Figure 1a: The Y axis legend should read “Number of chickens” instead

Answer: The legend in the Y axis was modified as “Number of chickens”. This modification was done also for Figure 2a and 3a.

• Comment 18. Line 196: it should read “a HPAI” instead of “an HPAI”, please check the test for similar mistakes and correct them all.

Answer: Thank you for this observation, the an as been changed as a in the sentence “which mimics the field situation of “a” HPAI outbreak“ and throughout the manuscript.

• Comment 19. Line 199: Perhaps the authors can replace “who” with “which all”, and dele “all” that comes after “had”.

Answer: The wording was changed according to your suggestion as follows “three chickens survived until 4 dpi, compared with those in the control group which all had died by 3 dpi”

• Comment 20. Lines 201-202: delete “of chickens”.

Answer: The word of chickens was deleted “Despite the mortality observed in the three groups, …”.

• Comment 21. Figure 2b: How were the statistics done here? Were the tissue titers from each group compared to the tissue titers across groups? Is the figure reflecting only a summary of the analysis results? If that was the case, please indicate so in figure legend. Please indicate so for each figure that involves statistical analysis.

Answer: Thank you for the question seeking more clarification. The statistical analyses were performed by comparing the average of same organ from four individuals in the treatment group (BXM and PR) to that of the control group. This clarification has been indicated in the caption of Figure 1b and 2b as follows “The viral titer calculated as the average (n=4) for each organ in the treatment group was compared to that of the control group”.

• Comment 22. Line 213: please replace “administration” with “administrations”

Answer: The s was added to the word administrations 

• Comment 23. Line 214: delete “the” at the beginning of the line and replace “of” with “with” before “HPAIV”.

Answer: the word “with” was added to HPAIV as follows “…complete protection in chickens against the lethal infection with HPAIV.”

• Comment 24. Line 220: move “treated” between “four” and “chickens”. Also, add “most of the” after “Viral replication in”.

Answer: Thank you for the suggestion, the sentence was modified as follows “However, the dosage of 0.1 and 0.5 mg/kg BXM only protected one of the four treated chickens (Figure 3a). Viral replication in the chicken organs was completely inhibited in chickens treated with 2.5, 12.5, and 62.5 mg/kg BXM (Figures 3e–g)”.

• Comment 25. Line 221. Please replace “low-titer virus was” with “low virus titers were”.

Answer:  In the sentence, the terminology was replaced as suggested “although low virus titer was detected in the lung and brain of two chickens treated with 2.5 mg/kg BXM (Figure 3e)

• Comment 26. Figure 3a: Perhaps the authors would like to select a different color scheme as all the treatment groups have similar colors.

Answer: Thank you for the observation, the color schemes in figure 3a was modified only for the negative control from red to green; for the others, our idea is to use the blue color for keeping the same meaning as Figure 1a and Figure 2a in which BXM is represented in blue. Here because of the different doses we used the blue color from the strong to the light one to indicated different doses.  

• Comment 27. Line 228: please add “of” after “Effects”.

Answer: the word “of” was added for well reading the sentence.

• Comment 28. Line 264: please add a period after mg/kg to start a new sentence. Also, add “ranging” after “titers”.

Answer: Thank you for the suggestion. The sentence was modified as follows “No virus was recovered from swabs of surviving chickens treated with 0.1, 0.5, 2.5, 12.5, and 62.5 mg/kg. However, high virus titers ranging from 2.7 to 4.2 log10 EID50/mL were recovered from the swabs collected on ….” 

• Comment 29. Line 265: you could add “the” after “on”.

Answer: “the” was added in the sentence “were recovered from the swabs collected on the three chickens that had died by 5 dpi in the 0.1 and 0.5 mg/kg …”

• Comment 30. Line 269: please replace “symptoms” with “signs”.

Answer: Symptom was replaced by signs as indicated in the sentence “…, viral excretion and clinical signs were predominantly suppressed by BXM activity at a dose of 2.5 mg/kg or higher”

• Comment 31. Line 289 and 290: replace “vaccine” with “vaccines” and add period after references for starting the new sentence.

Answer:  The modifications were made in the sentence as follows “Other studies have also evaluated the use of poultry vaccines for the prevention of HPAI caused by H5 and H7 virus strains in zoo birds [15,20,31]. However, an effective immune response was not induced in all of the vaccinated birds, …”

• Comment 32. Line 292-293: perhaps the sentence should read “Additionally, vaccines against AI are usually subtype- or strain-specific, whereas AI outbreaks are not predictable for such a vaccine specificity…”

Answer: Thank for the nice suggestion, the sentence was modified as accordingly “Additionally, vaccines against AI are usually subtype- or strain-specific, whereas the AI outbreak is not predictable for such a vaccine specificity

• Comment 33. Line 295: replace “this” with “the”.

Answer: In the following sentence, the word “this” was replaced by “the” as follows “In the present study, the chickens were used, the most susceptible avian species to AIV”

• Comment 34. Line 296: delete “the” before “chickens”. Also, “the most susceptible avian species to AIV” may not be completely true. Quails are even more susceptible than chickens. I would use "a susceptible species" instead

Answer: Thank you for the good observation about this statement, we have edited the sentence according to your suggestion as follows “…, chickens were used, a susceptible avian species to AIV, as an animal model to test for HPAIV infection…”

• Comment 35. Line 298: I would use “Our model demonstrates…” instead of “This demonstrates”.

Answer: We have really agreed with your suggestion and modified the statement as follows “Our model demonstrates that both BXM and PR were efficacious for the protection of chickens in simultaneous treatment”

• Comment 36. Line 304: add a period after chickens to start a new sentence.

Answer: Initially, a dose of 20 mg/kg of BXM or PR was administered to chickens. In the simultaneous treatment…

• Comment 37. Line 306: I would use “effects induced by BXM” instead of “effects for BXM”.

Answer: The word “for” was replaced with “by” in the sentence as follows “the survival of birds and virus titer in organs differed between the BXM and PR groups, with clear beneficial effects by BXM (Figures 1a and b)”.

• Comment 38. Line 323: replace “in” with “from”.

Answer: “in” has been replaced by “with” as indicated “No virus was recovered with swabs collected from chickens treated with 2.5 mg/kg of BXM”

• Comment 39. Lines 340-342: Please rephrase the sentence “Using the chicken and duck models, the administration of 2.5 mg/kg of BXM induced sufficient plasma concentration of BXA to protect birds from HPAI infection for 48 hours.”. The way it is written, it misleads to the conclusion that the birds were only protected for 48 hrs instead of referring to the period of time in which the drug's plasma levels were maintained.

Answer:  Thank you for the comment. Regarding the plasma concentration of BXA in both chickens and ducks, we referred to that in human and mouse reported sufficiently enough to suppress the virus replication. Because we did not assess such a low concentration detected behand 48 hpa with a challenge experiment, we could not suggest that this small amount of BXA can still protect birds from lethal infection. Reason why our conclusion indicates only the concentration at 48 hpa.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments on the manuscript " Evaluation of baloxavir marboxil and peramivir for the treatment of highly pathogenic avian influenza in chickens" (viruses-1015663). In this study, Twabela and colleagues investigated the use of two anti-influenza drugs baloxavir marboxil (BXM) and peramivir (PR) as anti-influenza drugs for the treatment of HPAI in chickens. This is a straightforward paper and their finding revealed that chicken treated immediately with 20 mg/kg of BXM twice a day for 5 days reduced virus replication and provided complete protection from infection compared with PR treated group. However, 24 hours delayed treatment, neither drugs showed incomplete protection against HPAI virus. Since HPAI causes highly lethal to chicken within a few days after the infection and hence the authors need to address the issue in using BXM (delayed treatment) in the context of HPAI-infection in poultry.  

Recently, Maki Kiso et al.,2019 (Viruses 2019, 11, 1066) and Mishin et al. 2019 (Emerg Infect Dis. 2019 Oct; 25(10) studied in vivo (mouse) and in vitro models well against Avian influenza viruses. The authors should be emphasized more, and the recent publications on the anti-viral efficacy of BXM and emergence of antiviral resistance of BXM (i.e PA-I38T/M/F) should be thoroughly discussed.

 A few minor comments:

Line 36-38: Rewrite or two separate sentences.

Line 56-59: Can be split into two sentences for more room to elaborate.

Line 296: Rewrite as …..avian species to AIV and so were used as…………  

Line 310-315: Can be summarized and shortened.

Author Response

Dear Editor and Reviewers,

We appreciate your great contribution to improving the quality of the manuscript we submitted in Viruses for publication. In the previous version, the manuscript was titled “Evaluation of baloxavir marboxil and peramivir for the treatment of highly pathogenic avian influenza in chickens” (ID Viruses-1015663) and it has been revised. In the current version, the title was slightly modified as “Evaluation of baloxavir marboxil and peramivir for the treatment of high pathogenicity avian influenza in chickens”, this has been changed according to the recent recommendation by the World Organisation for Animal Health (OIE).

All modifications according to the reviewers’ comments are made as tracking mode as per journal recommendation. Two new references have been inserted in line 62 and 343. In lines 61-64, the references have been permutated according to each statement.  Minor modifications were made in the Figures according to the reviewer’s comments.

Reviewer #2.

General comments: Recently, Maki Kiso et al.,2019 (Viruses 2019, 11, 1066) and Mishin et al. 2019 (Emerg Infect Dis. 2019 Oct; 25(10) studied in vivo (mouse) and in vitro models well against Avian influenza viruses. The authors should be emphasized more, and the recent publications on the anti-viral efficacy of BXM and emergence of antiviral resistance of BXM (i.e PA-I38T/M/F) should be thoroughly discussed.

Answer: Thank you for the observation concerning the possibility of strains resistant to BXM. We have included this point in the discussion part as follows “In our study, we used an avian origin HPAIV for the challenge experiment and the efficacy of BXM was demonstrated. We cannot minimize the possibility of the circulation of other avian influenza strains potentially resistant to BXM due to some mutations in PA protein as reported previously [30,36]”

Minor comments:

• Comment 1. Line 36-38: Rewrite or two separate sentences.

Answer: Thank you for the suggestion, the sentence has been rewrite and separated in two as follows “Highly pathogenic avian influenza (HPAI) is one of the most devastating avian viral diseases. The infection caused by the highly pathogenic avian influenza virus (HPAIV) induces an acute disease with high mortality reaching 100% in chickens [1,2]

• Comment 2. Line 56-59: Can be split into two sentences for more room to elaborate

Answer: The statement was split into two sentences for better understanding as follows “Anti-influenza drugs have been intensively developed for human treatment [16,17]. However, only few studies have assessed their effectivity on avian species; for instance, oseltamivir, a pro-drug for neuraminidase inhibition, has been evaluated for preventative and therapeutic effects against avian influenza virus (AIV) infection in poultry” 

• Comment 3. Line 296: Rewrite as …..avian species to AIV and so were used as…………

Answer:  Thank you for the comment, this same observation was done by another reviewer and we have modified the sentence as follows “In the present study, the chickens were used, a susceptible avian species to AIV, as an animal model to test for HPAIV infection….”

• Comment 4. Line 310-315: Can be summarized and shortened.

Answer: Thank you for the suggestion, the paragraph was revised as follows “These results indicate that BXM was more protective compared to PR; therefore, BXM was used for further experiments. The dosage of 20 mg/kg to chickens was higher compared to that used in humans [23] and mice; This dose was then adjusted to 2.5 mg/kg, which was determined to be the minimum dose that protected 100 % of infected chickens with complete inhibition of virus replication in the organs as well as virus shedding, although the virus was recovered at low titer in organs from two chickens in this treatment group (Figure 3e).”

Author Response File: Author Response.pdf