Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”

Xu, Ziqian; Fan, Na; Hou, Xin; Wang, Jing; Fu, Shihong; Song, Jingdong; Shi, Mang; Liang, Guodong

doi:10.3390/v13122422

Open AccessReply

Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”

by

Ziqian Xu

^1,†,

Na Fan

^1,†,

Xin Hou

^2,†,

Jing Wang

²,

Shihong Fu

¹,

Jingdong Song

¹,

Mang Shi

^2,* and

Guodong Liang

^1,*

¹

State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China

²

The Center for Infection and Immunity Study, School of Medicine, Sun Yat-sen University, Guangzhou 510006, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contribute equally to the work.

Viruses 2021, 13(12), 2422; https://doi.org/10.3390/v13122422

Submission received: 11 November 2021 / Revised: 29 November 2021 / Accepted: 30 November 2021 / Published: 3 December 2021

(This article belongs to the Special Issue Sand Fly-Borne Phleboviruses)

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Dear Professor Remi N. Charrel and Professor Jerome Depaquit, we thank you for your interest in our research and for your kind suggestions. We agree that the suggested approach for host identification, namely, PCR + NGS (or amplicon-based NGS), which reveals detailed taxonomic compositions from a pooled sample, is more powerful and informative in comparison to the one we had used (i.e., PCR + Sanger sequencing). Despite that, we carried out further analyses (described below), which confirmed that the species identification here is reliable, such that Phlebotomus chinensis is the most probable vector for Heidi virus (HEDV).

On the one hand, we carefully examined the DNA sequence trace chromatogram obtained from the Sanger sequencing performed in our study, which showed clear and unambiguous signals throughout except for a single position (position 457), which had a mixture of “A” and “T” (Figure 1). Since multiple mixed chromatography trace signals are expected with the presence of more than two species of sandflies, because they shared less than 90% nucleotide identity in the COI gene, we are highly confident to suggest that the majority, if not all, sandflies in the sample belonged to Ph. chinensis.

On the other hand, we performed taxonomy profiling of a (different) pooled sample containing HEDV, which was analyzed based on meta-transcriptomic NGS analysis (SRA accession: SRR16970469). Ph. chinensis samples were captured in Jul 2020 at Yuncheng of Shanxi Province. The sample collection was performed with light traps placed near a livestock shed that housed dogs and chickens. Species identification was carried out by an experienced field biologist, and subsequently, Ph. chinensis samples (n = 50) were pooled for total RNA sequencing. Although the sample characterized here was not the same as the one used in our previous study (none were left after the initial virus isolation procedures), it contained HEDV whose abundance level reached 56.4 Reads per million, or RPM, based on read mapping using bowtie 2 program version 2.3.5 [1], suggesting a relatively high viral load within the sample. Reads from meta-transcriptomics sequencing were then de novo assembled using megahit program version 1.2.8 [2] and compared against COI sequence databases using the BLAST program, which revealed three COI-related contigs associated with Ph. chinensis (1444.5 RPM), Bradysia spp. (8.6 RPM), and an unidentified member of the Order Diptera (45.1 RPM), respectively. Given the high abundance level of the HEDV, it is more likely to be associated with Ph. chinensis, the dominant species, than the other two species. Furthermore, an analysis of minor variants using Geneious software package version 11.04 [3], based on reads mapped to COI genes, did not suggest the presence of a second species within the sample. Although polymorphism was identified in 24 positions (Table 1), it is too low to imply the presence of another species. Collectively, Ph. chinensis is the dominant species within the HEDV-positive samples and the only sandfly species identified here.

Nevertheless, the meta-transcriptomics result here did indicate that the species composition of a pooled sample can be complex, as correctly pointed out by you. To resolve this issue, it requires species composition analyses from more HEDV positive samples, such that a more definite vector–virus relationship can be established. We are currently screening more sandfly pools with meta-transcriptomics as well as PCR + NGS approaches, and hopefully, this matter can be clarified in the future.

Author Contributions

Conceptualization, G.L. and M.S.; methodology, Z.X., S.F. and J.S.; software, Z.X., X.H. and M.S.; validation, Z.X., N.F. and S.F.; formal analysis, Z.X., N.F., X.H. and M.S.; investigation, Z.X., N.F. and J.W.; resources, S.F.; data curation, Z.X. and X.H.; writing—original draft preparation, M.S.; writing—review and editing, Z.X. and G.L.; visualization, Z.X. and X.H.; supervision, G.L.; project administration, S.F.; funding acquisition, M.S. and G.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the National Natural Science Foundation of China (81290342), the Development Grant of the State Key Laboratory of Infectious Disease Prevention and Control (2014SKLID103), Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project (2019ZT08Y464), and the United States National Institutes of Health U01 (AI151810).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All of the materials and data that were used or generated in this study are described and available in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

Langmead, B.; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 2012, 9, 357–359. [Google Scholar]
Li, D.; Liu, C.M.; Luo, R.; Sadakane, K.; Lam, T.W. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 2015, 31, 1674–1676. [Google Scholar]
Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar]

Figure 1. The trace chromatogram for Sanger sequencing of cytochrome c oxidase I (COI) gene within the HEDV positive sample. The amplicon is around 650 nucleotides in length. For each position, both forward and reverse base callings and chromatograms are presented, and their positions and consensus sequences are shown above the chromatograms.

Table 1. Minor nucleotide variants of Ph. chinensis COI gene in the sample containing HEDV.

Name	Position	Length	Change	Coverage	Polymorphism Type	Variant Frequency
C	1450	1	A -> C	3613	SNP (transversion)	7.70%
C	1299	1	T -> C	2515	SNP (transition)	12.20%
T	1219	1	C -> T	2198	SNP (transition)	13.80%
T	1215	1	C -> T	2193	SNP (transition)	11.40%
C	1152	1	T -> C	4251	SNP (transition)	13.30%
A	1134	1	G -> A	4705	SNP (transition)	18.50%
C	1107	1	T -> C	4541	SNP (transition)	10.30%
A	1023	1	G -> A	6631	SNP (transition)	27.80%
G	966	1	A -> G	6469	SNP (transition)	25.80%
C	831	1	T -> C	5120	SNP (transition)	15.30%
G	783	1	A -> G	5285	SNP (transition)	6.50%
T	675	1	C -> T	6392	SNP (transition)	24.40%
A	571	1	G -> A	10,628	SNP (transition)	9.00%
C	417	1	A -> C	5624	SNP (transversion)	15.60%
T	381	1	C -> T	4386	SNP (transition)	14.00%
T	366	1	C -> T	8409	SNP (transition)	6.90%
A	351	1	G -> A	8213	SNP (transition)	10.60%
G	255	1	A -> G	11,297	SNP (transition)	5.90%
G	129	1	A -> G	7800	SNP (transition)	7.80%
A	114	1	G -> A	6668	SNP (transition)	8.20%
T	96	1	A -> T	5738	SNP (transversion)	11.30%
C	91	1	T -> C	5643	SNP (transition)	8.60%
T	90	1	C -> T	5626	SNP (transition)	20.80%

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Xu, Z.; Fan, N.; Hou, X.; Wang, J.; Fu, S.; Song, J.; Shi, M.; Liang, G. Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”. Viruses 2021, 13, 2422. https://doi.org/10.3390/v13122422

AMA Style

Xu Z, Fan N, Hou X, Wang J, Fu S, Song J, Shi M, Liang G. Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”. Viruses. 2021; 13(12):2422. https://doi.org/10.3390/v13122422

Chicago/Turabian Style

Xu, Ziqian, Na Fan, Xin Hou, Jing Wang, Shihong Fu, Jingdong Song, Mang Shi, and Guodong Liang. 2021. "Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”" Viruses 13, no. 12: 2422. https://doi.org/10.3390/v13122422

APA Style

Xu, Z., Fan, N., Hou, X., Wang, J., Fu, S., Song, J., Shi, M., & Liang, G. (2021). Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”. Viruses, 13(12), 2422. https://doi.org/10.3390/v13122422

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Reply to Charrel, R.N.; Depaquit, J. Comment on “Xu et al. Isolation and Identification of a Novel Phlebovirus, Hedi Virus, from Sandflies Collected in China. Viruses 2021, 13, 772”

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