Complete genome sequence of Japanese encephalitis virus strain SDWF-2021 isolated from a Culex mosquito pool from a duck farm

ABSTRACT We report here the complete genome sequence of Japanese encephalitis virus (JEV) strain SDWF-2021, isolated from a Culex mosquito pool in a duck farm located in Shandong, China. The isolated JEV genetically belong to genotype I, which is the dominant genotype circulation in China.

J apanese encephalitis virus (JEV), belonging to genus Flavivirus of the family Flaviviridae, is an enveloped, positive-sense single-stranded RNA virus (1).JEV is an important zoonotic pathogen with a transmission cycle maintained by mosquito vectors (Culex mosquitoes) and vertebrate-amplifying hosts (birds and pigs) (2,3), and it causes encephalitis in humans and abortion and orchitis in breeding pigs (4,5).JEV is phylogenetically divided into five genotypes (GI to V) based on the nucleotide sequence of the E gene (2).
In July 2021, approximately 1,000 Culex mosquitoes were collected from a duck farm located in Hanting District, Weifang city of Shandong Province in China.Mosquitoes were pooled per 20, grounded in RNase-free water, and centrifuged at 4°C to collect the supernatant.RNAs in supernatant were extracted by the TIANamp Virus RNA Kit (TIANGEN, Beijing, China) and utilized for the detection of JEV by an established duplex TaqMan probe-based RT-qPCR assay (6).A total of 1/51 (1.96%) Culex mosquito pools gave positive result.Subsequently, the supernatant of JEV-positive sample was collected to seed onto monolayers of BHK-21 cells and incubated at 37°C, 5% CO 2 for 3 days.The supernatant of cells with typical cytopathic effects was collected to pass in BHK-21 cells up to five times.RNA was extracted from the fifth passage and reverse transcribed into cDNA using the Superscript IV first-strand synthesis system (ThermoFisher Scientific, Waltham, MA, USA), and four pairs of primers targeting conserved regions of the full-length sequences of GI and GIII JEV were designed to amplify the complete genome of SDWF-2021 (2).Meanwhile, the 5′ and 3′ terminal ends of viral genome were amplified using the SMART 5′ RACE and 3′ RACE (Takara, Kyoto, Japan) and a gene-specific primer (Table 1).A total of four overlapping fragments (F1, F2, F3, and F4) were amplified and cloned into the pEasy-blunt vector (TransGen Biotech, Beijing, China).Three or four clones from each PCR product were sequenced via Sanger's DNA sequencing using the primers based on conserved regions of the full-length sequences of GI JEV (Table 1).The sequences of four fragments were assembled into the complete genome of JEV SDWF-2021 strain, and the open reading frame was determined by comparison with the classical strain SA-14 (GenBank accession number KU323483.1).
The complete genome sequence of SDWF-2021 was 10,965 bp in length, and its G + C content was 51.72%.Based on the processing principles of flavivirus polyprotein (7), the polyprotein of JEV SDWF-2021 strain was composed of 10 proteins, including 3 structural  proteins (C, prM, and E) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).Nucleotide similarity analysis indicated that the complete genome sequence of SDWF-2021 displayed the nucleotide sequence identities of 88.8% and 89.1% to vaccine strain SA-14-14-2 (GenBank accession number JN604986.1)and classical strain SA-14, respectively.Phylogenetic comparison of the E gene sequence of SDWE-2021 to those of other JEV strains (Fig. 1) showed that SDWE-2021 belongs to GI, which is the dominant genotype circulation in China (6).To date, the data on the prevalence of GI JEV in mosquitoes are lacking in China.The sequences of JEV SDWF-2021 strain will facilitate future research on the epidemiology and evolutionary biology of GI JEV in China.

FIG 1
FIG 1 Phylogenetic analysis of JEVs based on nucleotide sequences of E gene.A total of 23 JEV strains including the strain isolated in this study (covering five genotypes), with clear background origin and known full genome sequences, were selected for phylogenetic analysis.The phylogenetic tree was constructed by the neighbor-joining method in MEGA 7.0 software (Kimura two-parameter model; 1,000 bootstrap replicates), yielding only values greater than 70%.The tree is drawn to scale, with branch lengths measured in the number of nucleotide substitutions per site.Five distinct sublineages were identified: GI, GII, GIII, GIV, and GV.Red dot indicates the JEV isolate (SDWF-2021) from the Culex mosquito pools.

TABLE 1
Primers used in this study a F refers to forward primer, and R refers to reverse primer.