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. 2022 Jun 29:12:874003.
doi: 10.3389/fcimb.2022.874003. eCollection 2022.

Multiple Novel Mosquito-Borne Zoonotic Viruses Revealed in Pangolin Virome

Duo Zhang  1 , Min Zheng  2 , Ying Zhang  3 , Guanrong Feng  1 , Chengcheng Peng  1 , Chenghui Li  4 , Yiquan Li  5 , He Zhang  6 , Nan Li  1 , Pengpeng Xiao  1
Affiliations

Multiple Novel Mosquito-Borne Zoonotic Viruses Revealed in Pangolin Virome

Duo Zhang et al. Front Cell Infect Microbiol. .

Abstract

Swab samples were collected from 34 pangolins in Guangxi Province, China. Metavirome sequencing and bioinformatics approaches were undertaken to determine the abundant viral sequences in the viromes. The results showed that the viral sequences belong to 24 virus taxonomic families. To verify the results, PCR combined with phylogenetic analysis was conducted. Some viral sequences including Japanese encephalitis virus (JEV), Getah virus (GETV), and chikungunya virus (CHIKV) were detected. On the basis of the metavirome analysis, seven segments belonging to JEV were further identified through PCR amplification. Sequence comparison showed that, among seven sequences, JEV-China/P2020E-1 displayed the highest nucleotide (80.6%), with the JEV isolated in South Korea, 1988, and all of which belonging to genotype III. Seven CHIKV sequences were detected, with the highest homology (80.6%) to the Aedes africanus in Côte d'Ivoire, 1993. Moreover, passage from BHK-21 to Vero cells makes the newly isolated CHIKV-China/P2020-1 more contagious. In addition, the newly verified GETV sequences shared 86.4% identity with the 1955 GETV isolated from Malaysia. Some sudden and recurrent viruses have also been observed from the virome of pangolin in Guangxi Province, China; hence, dissemination tests will be implemented in the future.

Keywords: metagenomic analysis; mosquito-borne virus; pangolin; phylogenetic analysis; viral isolation and identification.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Sample collection site in Guangxi Province, China (2020). The sample collection site was labeled with a solid red circle.
Figure 2
Figure 2
Abundance and relationship of the virus families in the three samples. (A) Classification of the viral sequences was according to the virus family, with the relative abundance shown in a heat map. (B) Number of virus families in the three samples laid out in a Venn diagram.
Figure 3
Figure 3
Phylogenetic trees based on JEV-E. The trees were constructed according to the alignments of the nucleotide sequences of the E gene of Japanese encephalitis virus (JEV). The maximum likelihood method was applied using MEGA 7.0, in which the bootstrap value was set to 1,000. Solid red circles denote the viral genes detected in this study.
Figure 4
Figure 4
Phylogenetic trees based on CHIKV-E1/NS1. The trees were constructed according to the alignments of the nucleotide sequences of the E1 gene (A) and the NS1 gene (B) of chikungunya virus (CHIKV). The maximum likelihood method was applied using MEGA 7.0, in which the bootstrap value was set to 1,000. Solid red circles denote the viral genes detected in this study.
Figure 5
Figure 5
Phylogenetic trees based on GETV-NS3. The trees were constructed according to the alignments of the nucleotide sequences of the NS3 protein of Ross River virus (RRV). The maximum likelihood method was applied using MEGA 7.0, in which the bootstrap value was set to 1,000. Solid red circles denote the viral genes detected in this study.
Figure 6
Figure 6
Identification of CHIKV-China/P2020-1 isolated in Nanning City, Guangxi Province. (A) The cytopathic effect (CPE) of CHIKV-China/P2020-1 was observed after infection in BHK-21 cells. (B) Identification of CHIKV-China/P2020-1 by PCR. (C) Identification of CHIKV-China/P2020-1 by Western blot. (D) Negative-stain electron microscopy of CHIKV-China/P2020-1 particles.
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