One Health Genomic Surveillance at Human–Animal Interfaces in Rural Ghana Reveals Underreported Viruses of Zoonotic and Economic Concern

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The researchers conducted surveillance in the pigs, dogs, cattle, and bats in Ghana. Serum samples were collected from the pigs, dogs, and cattle, while tissue organs were collected from the bats. RNA from the samples were extracted, followed by cDNA generation, and sequencing using Illumina platform. After metagenomic analysis, they found Porcine Parvovirus 3, Canine Parvovirus, Rotavirus A genotype R16, and Bovine hepacivirus subtype B.  

Here are some comments and suggestions:

Minor revisions:

Line 140 : N=166

Line 146 : 800 µL

Line 154 : 8 µL

 

Major revisions:

Line 270-271 : The PPV is caused by DNA virus. But in the Materials and Methods, the authors used RNA extraction method. How do you explain this finding?

Is there a confirmation of the finding by testing the sample using different method, such as serology or RT-PCR?

Did the authors go back to the farm and to the specific swine to collect different samples for this disease to confirm it?

This information should be discussed in the Results or Discussion section.

 

Line 357-360 : The same as in the PPV, the CPV is caused by DNA virus. With the use of RNA extraction method for nucleic acid extraction step of DNA virus, how do you explain this finding?

Is there a confirmation of the finding by testing the sample using different method, such as serology or RT-PCR?

Did the authors go back to collect more representative sample for the CPV to confirm it?

This information should be discussed in the Results or Discussion section.

 

Line 409 – 411 and 457 - 461: Did the Bovine hepacivirus and the Rotavirus A findings in the sample were confirmed using different detection method?

This information should be discussed in the Results or Discussion section.

 

Discussion section: If the confirmatory tests were not conducted to the pathogens found in the positive samples, it could be added as one of limitations in the study.

Author Response

Minor revisions:

Line 140 : N=166

Line 146 : 800 µL

Line 154 : 8 µL

We thank the reviewer for their attention to detail and have addressed these items.

Major revisions:

1. Line 270-271 : The PPV is caused by DNA virus. But in the Materials and Methods, the authors used RNA extraction method. How do you explain this finding? 

We thank the Reviewer for their comment. Although PPV is a DNA virus, the Zymo Quick-RNA Viral Kit (Zymo Research, Irvine, CA, USA) used in this study is capable of co-purifying both viral RNA and DNA. According to the manufacturer’s protocol (https://zymoresearch.eu/collections/quick-rna-viral-kits/products/quick-rna-viral-kit), the column chemistry binds total nucleic acids, including ssDNA viruses such as parvoviruses, and the kit specifically notes compatibility with parvoviral nucleic acid isolation (see screenshots below). Therefore, the detection of PPV3 and CPV2 using this extraction method is consistent with the kit’s intended capabilities (https://zymoresearch.eu/collections/quick-rna-viral-kits/products/quick-rna-viral-kit).

 

 

2. Is there a confirmation of the finding by testing the sample using different method, such as serology or RT-PCR? 

We thank the Reviewer for their comment. Due to limited sample availability and resource constraints, additional confirmatory testing, such as RT-PCR or serological assays, was not performed on the positive samples.

3. Did the authors go back to the farm and to the specific swine to collect different samples for this disease to confirm it? This information should be discussed in the Results or Discussion section.

We thank the Reviewer for their comment. Due to limited logistical capacity, available supplies, and labor resources during the study period, we were unable to revisit farms or resample the specific swine with positive viral detections to collect additional specimen types for confirmatory testing. We acknowledge that follow-up sampling could have provided additional information regarding viral persistence, tissue distribution, and confirmation of infection status. We have added this point as a study limitation and future research direction in the revised Discussion section.

4. Line 357-360 : The same as in the PPV, the CPV is caused by DNA virus. With the use of RNA extraction method for nucleic acid extraction step of DNA virus, how do you explain this finding? Is there a confirmation of the finding by testing the sample using different method, such as serology or RT-PCR? Did the authors go back to collect more representative sample for the CPV to confirm it? This information should be discussed in the Results or Discussion section.

We thank the Reviewer for their question and confirm the same response as above: the Zymo kit is compatible with isolating parvoviruses per the manufacturer’s instructions. No additional samples were collected from farm animals due to constrained resources. We have added this as a future direction. 

5. Line 409 – 411 and 457 - 461: Did the Bovine hepacivirus and the Rotavirus A findings in the sample were confirmed using different detection method? This information should be discussed in the Results or Discussion section.

We thank the Reviewer for their comment. The same laboratory workflow was used for the detection of PPV3, CPV, and BovHepV, including viral nucleic acid extraction from serum samples, library preparation, sequencing, and downstream bioinformatics analyses, as described in the Methods section. For RVA, viral nucleic acids were extracted from homogenized bat organ tissues prior to sequencing using the same extraction kit, sequencing platform, and bioinformatics pipeline. No additional confirmatory detection methods were performed for these viruses beyond the metagenomic sequencing workflow described in the study. 

6. Discussion section: If the confirmatory tests were not conducted to the pathogens found in the positive samples, it could be added as one of limitations in the study.

We thank the Reviewer for their comment and have added this as a limitation in the discussion. 

Reviewer 2 Report

Comments and Suggestions for Authors

This study collected 165 animals sampled across Ghana five regions，The first discovery Porcine Parvovirus 3, Canine Parvovirus, Rotavirus A genotype R16, and Bovine hepacivirus subtype B. This provides excellent epidemiological data for the current health of both humans and animals. However, there are a few minor issues that need to be addressed:

1. The serum samples may have lower sensitivity in detecting certain viruses (such as PPV3) compared to the tissue samples. The bat samples included spleen, liver, kidney, and intestinal tissues, but for cattle, pigs, and dogs, only serum samples were used. Why?
2. The mixed sample strategy may dilute the viral load, resulting in the missed detection of low-abundance viruses. How did the authors ensure that no missed detections occurred after the mixed samples?
3. The CPV-2c variant strain, with a Q370R mutation in its VP2 protein, has an impact on the vaccine but does it fail to provide protection?
4. The estimation of TMRCA suggests that the specific evolutionary rate should be provided in the supplementary materials.
5. The detection of RVA in bats is of great significance. What are the specific hazards and can it cross-species transmission occur?

Author Response

Reviewer 2

This study collected 165 animals sampled across Ghana five regions，The first discovery Porcine Parvovirus 3, Canine Parvovirus, Rotavirus A genotype R16, and Bovine hepacivirus subtype B. This provides excellent epidemiological data for the current health of both humans and animals. However, there are a few minor issues that need to be addressed:

1. The serum samples may have lower sensitivity in detecting certain viruses (such as PPV3) compared to the tissue samples. The bat samples included spleen, liver, kidney, and intestinal tissues, but for cattle, pigs, and dogs, only serum samples were used. Why?

We thank the Reviewer for their comment. The bat samples included spleen, liver, kidney, and intestinal tissues because the collected bats were euthanized in accordance with approved ethical protocols, allowing organ collection for downstream analyses. In contrast, livestock sampling was conducted directly with farmers in community settings, and we aimed to minimize animal harm and avoid imposing financial losses associated with sacrificing cattle or pigs. Therefore, only serum samples were collected from these animals. Similarly, dogs were sampled non-lethally within the community, and only serum samples were obtained. 

 

2. The mixed sample strategy may dilute the viral load, resulting in the missed detection of low-abundance viruses. How did the authors ensure that no missed detections occurred after the mixed samples?

We acknowledge that pooling samples can reduce sensitivity for the detection of low-abundance viruses due to dilution effects. However, given financial and logistical constraints, pooled metagenomic sequencing represents a widely used and cost-effective approach that enables the screening of a larger number of samples while maintaining sufficient sequencing depth. We recognize that pooling inherently carries the limitation that some low-titer viral targets may remain undetected, and therefore it is not possible to completely exclude missed detections. To mitigate this limitation, we aimed to maximize sequencing sensitivity by generating a high sequencing depth (~20 million reads per barcode), which is consistent with commonly applied metagenomic sequencing standards in the literature (D. Liu et al., 2021; J. Liu et al., 2022; Paoli et al., 2026). This was achieved using the Illumina NextSeq P2 Reagent Kit v3 (100 cycles).

3. The CPV-2c variant strain, with a Q370R mutation in its VP2 protein, has an impact on the vaccine but does it fail to provide protection?

We thank the Reviewer for their comment. We would like to clarify that the Q370R mutation in the VP2 protein was discussed in the context of potentially enhanced pathogenicity, as previously described for certain CPV-2b strains, and not specifically in relation to vaccine failure. We did not directly assess vaccine efficacy in this study.

The discussion regarding potential antigenic drift was instead related to amino acid position 426, which differs among CPV-2 variants and has been suggested to influence antigenicity and host antibody recognition. Changes at this position may alter antibody binding affinity relative to CPV-2a or CPV-2b strains; however, the impact on vaccine-mediated protection was not evaluated in the present study. We have clarified this distinction in the revised Discussion section and added the following statement:

“Position 426 differs among the three CPV strains and has been suggested to contribute to antigenic drift, potentially affecting host antibody recognition [76]. The implications of these mutations for vaccine efficacy and immune protection will need to be investigated in future studies.”

 

4. The estimation of TMRCA suggests that the specific evolutionary rate should be provided in the supplementary materials.

We thank the Reviewer for their suggestion and have added the evolutionary rate of each virus to Supplementary Table 5. 

 

5. The detection of RVA in bats is of great significance. What are the specific hazards and can it cross-species transmission occur?

We thank the Reviewer for recognizing the significance of our findings. RVA is a major cause of diarrheal disease in mammals and is one of the leading causes of severe childhood diarrhea worldwide, particularly in low- and middle-income countries. The detection of RVA in bats is important because bats may serve as reservoirs of genetically diverse RVA strains with the potential for viral reassortment and interspecies transmission.

In our study, the recovered RVA segment 1 sequence belonged to the bat-associated R16 genotype and clustered separately from known human-associated clades, providing no direct evidence of cross-species transmission to humans based on the available sequence data. However, because only one of the eleven genome segments was recovered, we cannot exclude the possibility that other genome segments may share closer similarity with human- or other mammalian-associated RVA strains. Additional characterization and recovery of the complete Ghanaian bat RVA genome will be important to better assess its evolutionary relationships and potential for cross-species transmission. Previous studies in have found genetic and evolutionary evidence strongly supporting bat-to-human spillovers in the Dominican Republic (Bourdett-Stanziola et al., 2021), Thailand (Komoto et al., 2021), and Argentina (Simsek et al., 2021). 

 

References

Bourdett-Stanziola, L., Centeno, E., Nordgren, J., Durant-Archibold, A. A., Ortega-Barria, E., & Bucardo, F. (2021). Potential Bat-like Rotavirus in Hospitalized Children with Diarrhea from the Dominican Republic | Asian Journal of Research in Infectious Diseases. Retrieved May 27, 2026, from https://journalajrid.com/index.php/AJRID/article/view/148

Komoto, S., Tacharoenmuang, R., Guntapong, R., Upachai, S., Singchai, P., Ide, T., Fukuda, S., Hatazawa, R., Sutthiwarakom, K., Kongjorn, S., Onvimala, N., Luechakham, T., Sriwanthana, B., Murata, T., Uppapong, B., & Taniguchi, K. (2021). Genomic characterization of a novel G3P[10] rotavirus strain from a diarrheic child in Thailand: Evidence for bat-to-human zoonotic transmission. Infection, Genetics and Evolution, 87, 104667. https://doi.org/10.1016/j.meegid.2020.104667

Liu, D., Zhou, H., Xu, T., Yang, Q., Mo, X., Shi, D., Ai, J., Zhang, J., Tao, Y., Wen, D., Tong, Y., Ren, L., Zhang, W., Xie, S., Chen, W., Xing, W., Zhao, J., Wu, Y., Meng, X., … Wang, Y. (2021). Multicenter assessment of shotgun metagenomics for pathogen detection. eBioMedicine, 74. https://doi.org/10.1016/j.ebiom.2021.103649

Liu, J., Wang, X., Xie, H., Zhong, Q., & Xia, Y. (2022). Analysis and evaluation of different sequencing depths from 5 to 20 million reads in shotgun metagenomic sequencing, with optimal minimum depth being recommended. Genome, 65(9), 491–504. https://doi.org/10.1139/gen-2021-0120

Paoli, J. E., Aung, O., Lilak, A. A., Maw, M. T., Cleary, N. G., Watto, E., Hassell, J., Win, Y. T., Thein, W. Z., Evans, T. S., Valitutto, M., Goldstein, T., Johnson, C. K., Mazet, J. A., Fleischer, R., VanTassel, N., Subramaniam, K., Anderson, B. D., von Fricken, M. E., … Murray, S. (2026). Detection of Wencheng shrew virus and cardiovirus from small mammals in Myanmar. Scientific Reports, 16(1), 8885. https://doi.org/10.1038/s41598-026-38406-w

Simsek, C., Corman, V. M., Everling, H. U., Lukashev, A. N., Rasche, A., Maganga, G. D., Binger, T., Jansen, D., Beller, L., Deboutte, W., Gloza-Rausch, F., Seebens-Hoyer, A., Yordanov, S., Sylverken, A., Oppong, S., Sarkodie, Y. A., Vallo, P., Leroy, E. M., Bourgarel, M., … Matthijnssens, J. (2021). At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions. mBio, 12(1), e02755-20. https://doi.org/10.1128/mBio.02755-20