Isolation and Characteristics of a Novel Aichivirus D from Yak

ABSTRACT Aichivirus D (AiV-D) is a newly emerging Kobuvirus detected in bovine and sheep, and information is limited regarding its biological significance and prevalence. This study aimed to explore both the prevalence and characteristics of AiV-D in yaks. From May to August 2021, 117 fecal samples were collected from yaks with diarrhea in three provinces of China’s Qinghai-Tibet Plateau, 15 of which were selected and pooled for metagenomic analysis. A high abundance of AiV-D sequences was obtained. Of the 117 diarrhea samples, 29 (24.8%) tested AiV-D–positive, including 33.3% (14/42) from Sichuan, 21.1% (8/38) from Qinghai, and 18.9% (7/37) from Tibet, respectively, suggesting a wide geographical distribution of the AiV-D in yaks in the Qinghai-Tibet Plateau. Furthermore, three AiV-D strains were successfully isolated using Vero cells. Significantly, the AiV-D strain could cause diarrhea, intestinal bleeding, and inflammation in yak calves via oral inoculation. The virus was distributed in the ileum, jejunum, duodenum, colon, cecum, and rectum. Based on phylogenetic analysis of the genome and capsid protein P1 (VP0, VP3, and VP1 genes), the yak AiV-D strains likely represent a novel genotype of AiV-D. On the whole, this study identified a novel genotype of AiV-D from yaks, which was successfully isolated, and confirmed that this virus is a diarrhea pathogen in yaks and has a wide geographical distribution in the Qinghai-Tibet Plateau. Our results expand the host range of AiV-D and the pathogen spectrum of yaks and have significant implications for diagnosing and controlling diarrhea in yaks. IMPORTANCE In this study, we identified and successfully isolated a novel genotype of AiV-D from yaks. Animal infection confirmed that this virus can cause diarrhea, intestinal bleeding, and inflammation in yak calves via oral inoculation. The virus was distributed in the ileum, jejunum, cecum, duodenum, colon, and rectum. All of these results have significant implications for diagnosing and controlling diarrhea in yaks. These novel AiV-D strains have a wide geographical distribution in yaks from the Qinghai-Tibet Plateau in China. In addition to expanding the host range of AiV-D and the pathogen spectrum of yaks, these findings can increase knowledge of the prevalence and diversity of AiV-D.

The genetic characterization of the work is complete, refined, and well-described. However, the other study portions are not as refined and resemble data in a preliminary character. For example, the "prevalence" study is biased (diarrheic samples only). Furthermore, it does not provide any rationale for the sampling design and size of the population in question. Similarly, the pathogenesis study in yaks used a limited number of animals, and the design can be improved. For example, there is no fecal shedding data during the study (either virus titer or CT). This reviewer also has concerns about the lack of details presented in the material and methods. The quality of the figures is not ideal. For example, it looks like an additional supplement was added to the cell culture media of the virus cells based on the color of the media in the figure. The FA definition is not good, and I am not sure about the IHC specificity based on the provided picture and the lack of negative control images.
Reviewer #2 (Comments for the Author): In this manuscript, a novel genotype of AiV D from yaks was identified by Yan et al, and it was confirmed to be a diarrhea pathogen of yak calves by animal infection and the re-isolation of AiV D. Investigation of the prevalence and characteristics of AiV D was conducted in this study, and the results indicated that this novel AiV D strains have been widely prevalent in yaks from the Qinghai-Tibet Plateau in China, expanding the host range of AiV D and the pathogen spectrum of yaks. In general, abundant data was generated and presented in this manuscript, which might enhance the knowledge of the prevalence and diversity of AiV D. However, some concerns need to be addressed as below: Major points: 1. Among the 117 diarrhea samples from yak, 29 were detected to be AiV D positive by RT-PCR (lines 97-98). However, complete P1 region sequences were obtained from the 14 AiV D positive samples (lines 103-104). How did you choose the 14 AiV D positive samples from all the 29 AiV D positive samples? 2. Lines 140-142, the sentence "The phylogenetic trees based on complete genomes and individual genes all showed that these strains were grouped into AiV D species, forming an independent branch (Fig.4, Supplementary Fig S6-S8)" is not correct, because the phylogenetic tree based on the complete nucleotide sequences of AiV L gene did not form an independent branch as showed in Fig S6. Additionally, the value of bootstrap under 60 is usually not displayed in phylogenetic tree. 3. Lines 164-165, authors described that "Infected yak calves showed watery diarrhea and depression at three dpi, and diarrhea was most severe at six dpi ( Supplementary Fig.S10)". However, Supplementary Fig.S10 just showed the severe diarrhea at three dpi without six dpi. Viral shedding of infected yaks in different days post-infection by RT-qPCR is suggested to be added. 4. Lines 170-171, "AiV D was detected in the lymph, duodenum, jejunum, ileum, cecum, colon, and rectum of the two infected yak calves (Supplementary Table S3)", while in lines 166-167, "gross pathological changes were observed in the obtained duodenum and jejunum, characterized by intestinal bleeding (Supplementary Fig.S11)", how about the gross pathological changes in ileum, cecum, colon, and rectum? 5. Lines 264-265, "15 samples were selected from three provinces and pooled for metagenomics analysis as previously described". 15 samples were selected, Please explain the criteria. Minor points: 1. There are many grammatical errors in this manuscript. The English should be well polished. 2. Abbreviations should be defined at first mention in the main body part, and then subsequently used throughout the manuscript, such as dpi and TCID50. 3. Line 122, "10-6.5, 10-6.8 and 10-7 TCID50/0.1mL" do authors mean "106.5, 106.8 and 107 TCID50/0.1mL"? 4. There are many errors in this manuscript that need to be corrected, such as line 143 "The", line 209 "caused cause", line 212 "naturally infected", line 272 "94 •C", line 299 "Genbank"...Please check the whole manuscript carefully.

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In this manuscript, a novel genotype of AiV D from yaks was identified by Yan et al, and it was confirmed to be a diarrhea pathogen of yak calves by animal infection and the re-isolation of AiV D. Investigation of the prevalence and characteristics of AiV D was conducted in this study, and the results indicated that this novel AiV D strains have been widely prevalent in yaks from the Qinghai-Tibet Plateau in China, expanding the host range of AiV D and the pathogen spectrum of yaks. In general, abundant data was generated and presented in this manuscript, which might enhance the knowledge of the prevalence and diversity of AiV D. However, some concerns need to be addressed as below: Major points: 2. Abbreviations should be defined at first mention in the main body part, and then subsequently used throughout the manuscript, such as dpi and TCID 50 .
4. There are many errors in this manuscript that need to be corrected, such as line 143 "The", line 209 "caused cause", line 212 "naturally infected", line 272 "94 •C", line

Dear Editor/Reviewers
Thank you for kindly reviewing the manuscript entitled "Isolation and characteristics of a novel Aichivirus D from yak" (Spectrum00099-23). We are grateful for the professional suggestions of the reviewers. The manuscript has been revised accordingly, and revisions are marked in red in the revised manuscript.

Reviewer #1:
The study aims to characterize the genome of a novel Kobuvirus found in yaks. Furthermore, the study seeks to understand the prevalence of this virus in China and the pathogenesis in mice and yak.
The genetic characterization of the work is complete, refined, and well-described. However, the other study portions are not as refined and resemble data in a preliminary character. For example, the "prevalence" study is biased (diarrheic samples only). (3) . We have added detailed information regarding the materials and methods in the revised manuscript, including the deep sequencing and the establishment of the qRT-PCR method.
The methods details of qRT-PCR are listed in the supplementary materials.
(4) . We did not add additional supplements to the culture medium, probably due to the contrast of the cell pictures which did not look good, we have replaced the cell pictures with better quality. The anti-KoV polyclonal antibody was prepared in our laboratory for IF, and the anti-AiV-D polyclonal antibody was prepared in our laboratory for IHC, and the negative control for IHC was also added to Fig.2. The relevant description was added in the revised manuscript.
(5) . The English language within whole manuscript was revised carefully, we have used editing service to polish the English language of the revised manuscript. 2. Lines 140-142, the sentence "The phylogenetic trees based on complete genomes and individual genes all showed that these strains were grouped into AiV D species, forming an independent branch (Fig.4, Supplementary Fig S6-S8)" is not correct, because the phylogenetic tree based on the complete nucleotide sequences of AiV L gene did not form an independent branch as showed in Fig S6. Additionally, the value of bootstrap under 60 is usually not displayed in phylogenetic tree.
Response: Thank you for your suggestions, we have checked the phylogenetic tree and removed the wrong phylogenetic tree. The related sentences have been rewritten in the revised manuscript.
3. Lines 164-165, authors described that "Infected yak calves showed watery diarrhea and depression at three dpi, and diarrhea was most severe at six dpi ( Supplementary Fig.S10)".
However, Supplementary Fig.S10 just showed the severe diarrhea at three dpi without six dpi.
Viral shedding of infected yaks in different days post-infection by RT-qPCR is suggested to be added.

Response:
We are sorry for the incorrect description of the figure, the previous figure showed diarrhea at 6 dpi. We added 3 dpi and 6 dpi to the revised figure in Supplementary Fig.S7 in the revised manuscript. The fecal shedding of AiV-D in yaks was added in the revised manuscript, the methods details of qRT-PCR are listed in the supplementary materials.
4. Lines 170-171, "AiV D was detected in the lymph, duodenum, jejunum, ileum, cecum, colon, and rectum of the two infected yak calves (Supplementary Table S3)", while in lines 166-167, "gross pathological changes were observed in the obtained duodenum and jejunum, characterized by intestinal bleeding (Supplementary Fig.S11)", how about the gross pathological changes in ileum, cecum, colon, and rectum?

Response:
The description of pathological changes and the histopathological changes in the ileum, cecum, colon, and rectum were added to the result in the revised manuscript. 5. Lines 264-265, "15 samples were selected from three provinces and pooled for metagenomics analysis as previously described". 15 samples were selected, Please explain the criteria.

Response:
We are sorry that we did not describe clearly the criteria of selected samples for metagenomics analysis. The samples for deep sequencing were taken from 7 farms, except for 1 farm where 3 samples were randomly selected, and the remaining 6 farms where 2 samples were randomly selected from each farm, for 15 samples pooled for metagenomic analysis.
The relevant description has been rewritten in the revised manuscript. The detailed number of samples in each farm was also added to Table 1. Minor points: