Diagnosis of a Rabbit Hemorrhagic Disease Virus 2 (RHDV2) and the Humoral Immune Protection Effect of VP60 Vaccine
, Kaimin Song 1,†, Yongzhen Du 1, Zhuanglong Zhang 2, Rupeng Fan 1, Pimiao Zheng 2,* and Jianzhu Liu 1,*Round 1
Reviewer 1 Report
Rabbit hemorrhagic disease (RHD), a highly contagious and fatal disease of wild and domestic rabbits (Oryctolagus cuniculus), was first reported in China in 1984. Despite the development of vaccines, RHD cases are still reported. Perhaps this is due to the limited use of vaccines in some countries, as well as the genetic variability of the virus. The purposes of this work are clinical and pathological examinations on a rabbit farm in Tai`an, genetic characterization of the new RHDV isolate, study of the effectiveness of a new commercialized rabbit plague baculovirus vector (VP60) vaccine against RHDV2.
1. The main note is that it has been repeatedly shown that the VP60 protein expressed using the baculovirus vector can be used as vaccines for the prevention of RHD. Due to this fact, the novelty of this study is not very clear. If this is a comparative study of two commercial vaccines currently in use, then this study would need to be complemented. For example, compare the duration of the immune response.
2. If the new RHDV2 vaccine is a VLP-based vaccine, then the VLP may need to be shown by electron microscopy.
3. It is necessary to add more information (in the introduction or discussion) about previous studies on the expression of RHDV/RHDV2 VP60 using baculovirus vector, since not all of them are indicated in the references. In addition, it would be possible to add to the discussion the examples of other expression systems of this protein, their disadvantages or advantages in comparison with the baculovirus vector.
4. It may be necessary to add information on mortality and morbidity on the farm to the text of the manuscript. It seems to me that this is important information about the pathogenicity of the isolate.
5. Do I understand correctly that the primers indicated in Table 1 were used not only for sequencing, but also for diagnostic studies?
6. Have studies been conducted on pathological and histopathological changes in vaccinated rabbits after challenge? Have you tested blood and organ samples from immunized animals after challenge by RT-PCR or haemagglutination test to confirm that the samples do not contain the virus?
7. Have there been recent outbreaks of RHDV (classic subtype or RHDVa) in China? Understanding the circulating strains in a country is very important for vaccine selection.
Author Response
Dear Reviewer,
I am submitting a revised manuscript to you with “Diagnosis of a Rabbit Hemorrhagic Disease Virus 2 (RHDV2) disease and the humoral immune protection effect of VP60 Vaccine” (cimb-2478680).
Thanks for the reviewers’ comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. Below are our responses to the editors' and reviewers' comments.
We have accommodated your comments in the revised sections and marked with highlighted changes in yellow in the revised manuscript. We hope that the present form of the manuscripts will satisfy the requirements for publication. We look forward to hearing from you soon.
The responses to the reviewer’s comments are as follows:
Point-by-point response
With kind regards,
Professor Jianzhu Liu
#These authors contributed equally to this work.
* Corresponding author,
Dr. Jianzhu Liu (Author ID: 55794434900, ORCID: 0000-0003-3634-0712)
College of Veterinary Medicine,
Shandong Agricultural University, Tai`an 271018, China
Tel.: 0086-538-8246287; Fax: 0086-538-8241419
Email address: [email protected]
Rabbit hemorrhagic disease (RHD), a highly contagious and fatal disease of wild and domestic rabbits (Oryctolagus cuniculus), was first reported in China in 1984. Despite the development of vaccines, RHD cases are still reported. Perhaps this is due to the limited use of vaccines in some countries, as well as the genetic variability of the virus. The purposes of this work are clinical and pathological examinations on a rabbit farm in Tai`an, genetic characterization of the new RHDV isolate, study of the effectiveness of a new commercialized rabbit plague baculovirus vector (VP60) vaccine against RHDV2.
- The main note is that it has been repeatedly shown that the VP60 protein expressed using the baculovirus vector can be used as vaccines for the prevention of RHD. Due to this fact, the novelty of this study is not very clear. If this is a comparative study of two commercial vaccines currently in use, then this study would need to be complemented. For example, compare the duration of the immune response.
Response: Thank you very much for your affirmation and suggestion for our work. Regarding the use of the two commercial vaccines, before the outbreak of RHDV2 at a rabbit facility in Tai`an, we had already immunized the rabbits with the conventional vaccine, but they still contracted the disease. Subsequently, we used the new RHDV vaccine, and there were no further outbreaks at the facility. Therefore, we believe that the immunization efficacy of the new RHDV vaccine is superior to that of the classic vaccine. As for your question regarding the comparison of the duration of an immune response, we have not conducted any relevant studies yet. However, your question is very beneficial to our future research, and we will consider incorporating it into our upcoming research progress. Additionally, in the Discussion section, we have added the description "In order to conduct a more comprehensive study on the performance of the new vaccine, in future studies, we will incorporate relevant experiments to compare the immune response duration of the two vaccines and transmission electron microscopy experiments on the protein related to the new vaccine." (in revised manuscript in Line 347-351)
- If the new RHDV2 vaccine is a VLP-based vaccine, then the VLP may need to be shown by electron microscopy.
Response: Thank you very much for your suggestion of our work. The new RHDV2 vaccine is a commercialized vaccine, and the information provided in the commercial kit for purchasing the vaccine indicates a VLP-based vaccine without accompanying electron microscope images of recombinant VLPs. However, your suggestion is crucial for our future research. Therefore, in the Discussion section, we have also included that "In order to conduct a more comprehensive study on the performance of the new vaccine, in future studies, we will incorporate relevant experiments to compare the immune response duration of the two vaccines and transmission electron microscopy experiments on the protein related to the new vaccine." (in revised manuscript in Line 347-351)
- It is necessary to add more information (in the introduction or discussion) about previous studies on the expression of RHDV/RHDV2 VP60 using baculovirus vector, since not all of them are indicated in the references. In addition, it would be possible to add to the discussion the examples of other expression systems of this protein, their disadvantages or advantages in comparison with the baculovirus vector.
Response: Thank you very much for your affirmation and suggestion for our work. We have added more information (in the Discussion section of the revised manuscript) about previous studies on the expression of RHDV/RHDV2 VP60 using a baculovirus vector.
Line 328-333: Wang et al [48]. used recombinant Lactobacillus casei expressing vp60 of RHDV to produce a vaccine that can be immunised orally, was safe and effective in inducing mucosal immunity. Omar Farnós et al [49]. used the high expression capacity of Pichia yeast to recombinantly express RHDV vp60 to protect mice from RHDV. Qi et al. [37] used Baculovirus expression vectors to express vp60 of RHDV and RHDV2 and produced a bivalent vaccine that protects rabbits against RHDV.
Line 339-341: Baculovirus expression vectors are easy to culture, express proteins, and mature in mass production, so they are widely used in laboratories or factories to produce VLP [51].
- It may be necessary to add information on mortality and morbidity on the farm to the text of the manuscript. It seems to me that this is important information about the pathogenicity of the isolate.
Response: Thank you very much for your affirmation and suggestion for our work. We have added information on mortality and morbidity on the farm to the revised manuscript.
Line101-105: Approximately 1500 rabbits were present in a rabbit farm in Tai’an, Shandong Province prior to the disease outbreak. Within ten days of the outbreak, 744 rabbits died, resulting in an average mortality rate of 49.6%. The rabbit farm promptly followed the regulations stipulated by the Animal Epidemic Prevention Law of China for the disposal of the affected rabbits during the outbreak.
- Do I understand correctly that the primers indicated in Table 1 were used not only for sequencing, but also for diagnostic studies?
Response: Thank you very much. Your understanding is correct. The primers in Table 1 can be used for sequencing as well as for clinical diagnostics.
- Have studies been conducted on pathological and histopathological changes in vaccinated rabbits after challenge? Have you tested blood and organ samples from immunized animals after challenge by RT-PCR or haemagglutination test to confirm that the samples do not contain the virus?
Response: Thank you very much for your question. Because the rabbits that received the new vaccine did not show any symptoms after the challenge, we did not perform a necropsy on the healthy rabbits. Furthermore, the rabbits that received the vaccine remained healthy compared to the control group, and there were no significant pathological or histological changes. In addition, the healthy rabbits that did not develop any symptoms did not show any evidence of RHDV2 spread in their blood and organ tissues. We have taken your suggestion into consideration and have included the relevant descriptions in the revised manuscript’s Results section.
Line 264-266: Compared with normal rabbits, vaccinated rabbits did not show changes in pathology and histopathology after the challenge, and RT-PCR did not detect the spread of RHDV2 in blood and tissue organs.
- Have there been recent outbreaks of RHDV (classic subtype or RHDVa) in China? Understanding the circulating strains in a country is very important for vaccine selection.
Response: Thank you very much for your question. According to relevant reports, in April 2020, an outbreak of RHDV2 occurred in Jin Tang County, Sichuan Province, China, resulting in an average mortality rate of 42.85%. As a result, over 3,500 rabbits were culled and destroyed, leading to significant economic losses for farmers. In 2021, there were reports of acute deaths among rabbits in various regions, including Henan and Shandong, which were confirmed to be caused by RHDV2. This indicates that RHDV2 spreads rapidly and highlights the lack of effective vaccines to prevent its transmission. In contrast, there is a well-established vaccine and control system for RHDV, although it is currently limited to a small area of spread.
Reviewer 2 Report
Dear authors,
I believe that your work satisfactorily describes the control of the negative effects of RHD infections on farm’s rabbits using a recombinant vaccine based in the VP60 gene of RHDV2 variant cloned in a commercialized rabbit plague baculovirus vector.
Your article is satisfactory for its inclusion in the MDPI CIMB journal after some minor changes and the answer to an additional question related with the experimental design as major comment.
Major comments:
Can you clarify why you didn’t include the rabbits immunized with the classic vaccine and the classic + new vaccine in the challenge against RHDV2, specially because all the other parameters related with the vaccination have been included in the document? Although you describe some significant differences between experimental groups, your experimental results point the possible protection of the classic vaccine against the RHDV2 challenge.
Minor comments:
Line 33: Please, could you change “the highest” by “higher”?
Line 65: Please, could you introduce a new line after the point before “RHDV is an icosahedral…”? I believe that the paragraph that begins with this sentence is clearly different from the precedent.
Line 145: Please, could you change “Eosin” by “eosin”?
Line 220: Please, could you change “branch” by “cluster”?
Line 329: Please, could you change “attack” by “challenge”?
Author Response
Dear Reviewer,
I am submitting a revised manuscript to you with “Diagnosis of a Rabbit Hemorrhagic Disease Virus 2 (RHDV2) disease and the humoral immune protection effect of VP60 Vaccine” (cimb-2478680).
Thanks for the reviewers’ comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. Below are our responses to the editors' and reviewers' comments.
We have accommodated your comments in the revised sections and marked with highlighted changes in yellow in the revised manuscript. We hope that the present form of the manuscripts will satisfy the requirements for publication. We look forward to hearing from you soon.
The responses to the reviewer’s comments are as follows:
Point-by-point response
With kind regards,
Professor Jianzhu Liu
#These authors contributed equally to this work.
* Corresponding author,
Dr. Jianzhu Liu (Author ID: 55794434900, ORCID: 0000-0003-3634-0712)
College of Veterinary Medicine,
Shandong Agricultural University, Tai`an 271018, China
Tel.: 0086-538-8246287; Fax: 0086-538-8241419
Email address: [email protected]
I believe that your work satisfactorily describes the control of the negative effects of RHD infections on farm’s rabbits using a recombinant vaccine based in the VP60 gene of RHDV2 variant cloned in a commercialized rabbit plague baculovirus vector.
Your article is satisfactory for its inclusion in the MDPI CIMB journal after some minor changes and the answer to an additional question related with the experimental design as major comment.
Major comments:
1. Can you clarify why you didn’t include the rabbits immunized with the classic vaccine and the classic + new vaccine in the challenge against RHDV2, specially because all the other parameters related with the vaccination have been included in the document? Although you describe some significant differences between experimental groups, your experimental results point the possible protection of the classic vaccine against the RHDV2 challenge.
Response: Thank you very much for your affirmation and suggestion for our work. Before the outbreak of RHDV2 in the rabbit farm, all the rabbits had been vaccinated with the classic vaccine. However, they still contracted the disease, leading us to believe that the classic vaccine does not provide protection against RHDV2, which is why the outbreak occurred. On the other hand, after administering the new RHDV2 vaccine and conducting a challenge test, the rabbits did not develop the disease, confirming the protective effect of the new vaccine against RHDV2. It is important to note that RHDV and RHDV2 have different antigenic properties. Therefore, in the challenge experiment, we did not expose the rabbits from the classic vaccine group and the classic vaccine + new vaccine group to the virus.
Minor comments:
- Line 33: Please, could you change “the highest” by “higher”?
Response: Thank you very much. We have changed “the highest” to “higher” in Line 33 in the revised manuscript.
- Line 65: Please, could you introduce a new line after the point before “RHDV is an icosahedral…”? I believe that the paragraph that begins with this sentence is clearly different from the precedent.
Response: Thank you very much for your suggestion. We have added a new line after the point before “RHDV is an icosahedral…” in Line 65-66 in the revised manuscript.
- Line 145: Please, could you change “Eosin” by “eosin”?
Response: Thank you very much for your suggestion. We have changed the “Eosin” to “eosin” in Line 149 in revised manuscript.
- Line 220: Please, could you change “branch” by “cluster”?
Response: Thank you very much for your suggestion. We have changed the “branch” to “cluster” in Line 224 in revised manuscript.
- Line 329: Please, could you change “attack” by “challenge”?
Response: Thank you very much for your suggestion. We have changed the “attack” to “challenge” in Line 342 in revised manuscript.
Round 2
Reviewer 1 Report
This study is interesting and relevant. In my opinion, the changes made help to better understand the objectives of the study and demonstrate the results of the study.
