Metavirome Analysis and Identification of Midge-Borne Viruses from Yunnan Province, China, in 2021

Abstract

Midges are widely distributed globally and can transmit various human and animal diseases through blood-sucking. As part of this study, 259,300 midges were collected from four districts in Yunnan province, China, to detect the viral richness and diversity using metavirome analysis techniques. As many as 26 virus families were detected, and the partial sequences of bluetongue virus (BTV), dengue virus (DENV), and Getah virus (GETV) were identified by phylogenetic analysis and PCR amplification. Two BTV gene fragments, 866 bps for the VP2 gene of BTV type 16 and 655 bps for the VP5 gene of BTV type 21, were amplified. The nucleotide sequence identities of the two amplified BTV fragments were 94.46% and 98.81%, respectively, with two classical BTV-16 (GenBank: JN671907) and BTV-21 strains (GenBank: MK250961) isolated in Yunnan province. Furthermore, the BTV-16 DH2021 strain was successfully isolated in C6/36 cells, and the peak value of the copy number reached 3.13 × 10⁷ copies/μL after five consecutive BHK-21 cell passages. Moreover, two 2054 bps fragments including the E gene of DENV genotype Asia II were amplified and shared the highest identity with the DENV strain isolated in New Guinea in 1944. A length of 656 bps GETV gene sequence encoded the partial capsid protein, and it shared the highest identity of 99.68% with the GETV isolated from Shandong province, China, in 2017. Overall, this study emphasizes the importance of implementing prevention and control strategies for viral diseases transmitted by midges in China.

Keywords: metavirome analyses; midge-borne viruses; phylogenetic analyses; virus identification; virus isolation.

Figure 1

Phylogenetic tree based on the bluetongue virus type 16 (BTV-16) VP2 gene. (a) The application of the maximum likelihood method was used in Mega 7.0, where the bootstrap value was set to 1000. The solid red circle was used to mark the viral gene amplified in this study. (b) The VP2/VP5 amino acid sequences of BTV-16 DH2021/BTV-21 ML2021 were compared with the BTV-16 China/Yunnan 2017 strain.

Figure 2

Phylogenetic tree based on the dengue virus (DENV) E gene. The application of the maximum likelihood method was used in Mega 7.0, where the bootstrap value was set to 1000. The solid red circle was used to mark the partial viral gene segments amplified in this study.

Figure 3

Phylogenetic tree based on the Getah virus (GETV) capsid protein gene. The application of the maximum likelihood method was used in Mega 7.0, where the bootstrap value was set to 1000. The solid red circle was used to mark the partial viral gene segments amplified in this study.

Figure 4

Isolation and identification of the BTV-16 DH2021 strain. (a) An obvious cytopathic effect (CPE) could be observed on the C6/36 cells (100×). (b) Identification of the effective expression of the BTV target protein using Western blot analysis. (c) BTV-16 DH2021 strain viral particles with a diameter of approximately 70 nm were visualized using negative stain electron microscopy. The scale of the original image is 200 nm. An enlarged view of a BTV particle is shown in the upper right corner. (d) The virus growth curve of BTV-16 DH2021 strain was determined within 72 h.

Figure 5

Regional distribution of midge samples collected in Yunnan province, China (orange-red). In total, 61,000 midges were collected in Dehong (coordinates: 24°43′22″ N 97°34′16″ E A: 261.0 m), 75,000 midges (coordinates: 25°24′26″ N 101°14′30″ E A: 1999.8 m) were collected in Chuxiong, 65,000 midges were collected in Xishuangbanna (coordinates: 21°26′17″ N 101°19′18″ E A: 575.0 m), and 58,300 midges were collected in Lincang (coordinates: 23°29′6″ N 98°50′5″ E A: 472.0 m).