First Molecular Detection and Genetic Analysis of a Novel Porcine Circovirus (Porcine Circovirus 4) in Dogs in the World

ABSTRACT A novel circovirus species was identified in farmed pigs and designated porcine circovirus 4 (PCV4); it has recently been proved to be pathogenic to piglets. However, little is known about its cross-species transmission, and there is no evidence of PCV4 in dogs. A total of 217 fecal samples were collected from diarrheal dogs in Henan Province, China, and tested for the presence of PCV4 using a real-time PCR assay. Among the 217 samples, the total positivity rate for PCV4 was 5.99% (13/217 samples), with rates of 7.44% and 4.17% in 2020 and 2021, respectively. PCV4 was detected in dogs in 6 of 10 cities, demonstrating that PCV4 could be detected in dogs in Henan Province, China. One PCV4 strain (HN-Dog) was sequenced in this study and shared high levels of identity (97.9% to 99.6%) with reference strains at the genome level. Phylogenetic analysis based on complete genome sequences of HN-Dog and 42 reference strains showed that the HN-Dog strain was closely related to 3 PCV4 reference strains (from pig, raccoon dog, and fox) but differed genetically from other viruses in the genus Circovirus. Three genotypes, i.e., PCV4a, PCV4b, and PCV4c, were confirmed by phylogenetic analysis of complete genome sequences of 42 PCV4 strains, and one amino acid variation in Rep protein (V239L) and three amino acid variations in Cap protein (N27S, R28G, and M212L) were considered conserved genotype-specific molecular markers. In conclusion, the present study is the first to report the discovery of the PCV4 genome in dogs, and the association between PCV4 infection and diarrhea warrants further study. IMPORTANCE This study is the first to report the presence of PCV4 in dogs worldwide, and the first complete genome sequence was obtained from a dog affected with diarrhea. Three genotypes of PCV4 strains (PCV4a, PCV4b, and PCV4c) were determined, as supported by specific amino acid markers (V239L for open reading frame 1 [ORF1] and N27S R28G and M212L for ORF2). These findings help us understand the current status of intestinal infections in pet dogs in Henan Province, China, and also prompted us to accelerate research on the pathogenesis, epidemiology, and cross-species transmission of PCV4.

2. This study included sample collection from symptomatic dogs, and there was no control group, one major limitation of the current. Without an appropriate control group, it is impossible establish a casual relationship between PCV4 and diarrhea in dogs and to exclude the possibility that PCV4 detected were simply by-standers, especially co-infection rate of PCV4 with other canine diarrheal viral agents was high (85%, 11/13).
3. Thirteen dog stool samples were PCV4 DNA positive but only one complete genome was derived. Was it due to low viral load of other samples? 4. Line 243: The sybr green-based PCR method they cited can simultaneously detected both PCV2 and PCV4. As the author only sequenced complete genome of one dog PCV4 derived from the current study and presented no additional data on phylogenetic analysis of partial sequences of the rest of PCV4 PCR+ ve samples (n=12), the PCV4 specificity is not strong. This requires further elaboration or additional experimental confirmation. 5. The complete genome was sequenced by first amplifying overlapping regions using GreenTaq, followed by cloning into plasmid and then Sanger sequencing. GreenTaq appears to be not a proofreading DNA polymerase and using it for clonal sequencing was problematic, so Taq-introduced random mutation would be sequenced. This is of concern as the authors attempted to link biological meaning of mutations detected in Cap and Rep genes. At least the authors need to confirm the genome sequence using consensus amplicon sequencing prepared by high-fidelity DNA polymerase using a rolling circle method for circular DNA genome.
6. To eliminate the possibility of PCV4 detection in dogs due to environmental contamination, it would be useful to provide more information whether pigs were also treated in the animal hospitals and whether the molecular laboratory handled pig samples. 7. Line 254: What does "thermistor parameter" mean? 8. Line 262: Specify it was Sanger sequencing 9. Lines 263-267: MEGA 6 or 7 was used?
10. Phylogenetic tree: Suggest adding labels to indicate PCV types 1-4. 11. Table 3: Suggest including the purpose of the three pairs of primers. Were they for complete genome sequencing only?
12. The discussion part end abruptly with the description of rep and cap mutation. A separate, concluding paragraph should be added to sum up take home messages of the current study.

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Manuscript Number (Spectrum04333-22R1): First molecular detection and genetic analysis of the novel porcine circovirus (PCV4) in dogs with diarrhea disease

Dear Editor and Reviewers,
Thank you for giving us the opportunity to re-submit our revised manuscript. We appreciate the concerns and suggestions provided by the reviewers and editor, and we have revised our manuscript accordingly. The modified parts in the text were indicated with red colour, and the responses to the reviewers are as following. These suggestions helped us improve the manuscript, and we hope that you find it suitable for publication.
I look forward to hearing from you soon. Response: Thanks for your question. In fact, the samples were originally used to test for diarrhea-associated viruses in dogs. Considering that PCV2 and PCV3 can be detected in dogs, we then attempted to detect PCV4 in these clinical samples.
Surprisingly, PCV4 DNA was detected in these diarrhea samples. However, the prevalence of PCV4 in healthy animals was unknown and warranted further study. We also apologized for our inappropriate extrapolation that established a casual relationship between PCV4 and diarrhea in dogs, which has been revised and marked in red in the manuscript. In the future we will also try your suggestion. Samples from healthy dogs will be collected as controls for PCV4 testing.

Comment 3: Thirteen dog stool samples were PCV4 DNA positive but only one complete genome was derived. Was it due to low viral load of other samples?
Response: Thanks for your question. The reasons why only one sequence is obtained were as follows: First, the viral load of some samples was low. Second, the genome of PCV4 has a stem-loop structure that makes it difficult to amplify. Finally, four wholegenome sequences were obtained during the experiment, but they shared 100% identity.
Then, they were not mentioned again in the manuscript. Response: We appreciate your constructive comments. In order to verify the specificity of the method, the amplification products of the detection primer pairs were submitted to Sangon Biotech Shanghai Co, Ltd., China for sequencing. Sequence alignment showed that the amplification results of the test primers belong to the PCV4 genome.
The reason why only one whole genome was analyzed is shown in the "Response to Comment 3". Comment 5: The complete genome was sequenced by first amplifying overlapping regions using GreenTaq, followed by cloning into plasmid and then Sanger sequencing. GreenTaq appears to be not a proofreading DNA polymerase and using it for clonal sequencing was problematic, so Taq-introduced random mutation would be sequenced. This is of concern as the authors attempted to link biological meaning of mutations detected in Cap and Rep genes. At least the authors need to confirm the genome sequence using consensus amplicon sequencing prepared by high-fidelity DNA polymerase using a rolling circle method for circular DNA genome.
Response: Thanks for your suggestion. According to your suggestion, PrimeSTAR® Max DNA Polymerase (Takara, Dalian, China), a high-fidelity DNA polymerase, was used to amplify the whole genome of PCV4 through three independent, overlapping DNA fragments. Sequence alignment showed that the amplification results were consistent with previous result. The most likely reason could be that three positive clones containing recombinant plasmids were independently sequenced, and no base errors occurred during sequence amplification. Thanks again for your suggestion. In the future, we will use high-fidelity enzymes for genome amplification. Response: Thanks for your suggestion. Labels denoting PCV types 1-4 have been added. In Fig. 2A, red solid circle (•), red solid triangle (▲) and red solid square (■) represent porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2) and porcine circovirus 3 (PCV3), respectively. Comment 11: Table 3: Suggest including the purpose of the three pairs of primers.
Were they for complete genome sequencing only? Response: Thanks for your suggestion. The three pairs of primers in Table 3 were used for complete genome sequencing only. In addition, a pair of detection primers was added in Table 3. Moreover, we annotate the purpose of primer pairs in Table 3. Response: Thanks for your suggestion. A separate, concluding paragraph has been added to sum up take home messages of the current study.