Comparative analysis of duck Tembusu virus Cluster 1 and Cluster 2.1 in Culex tritaeniorhynchus: Insights into viral characteristics, infectivity, and innate immune response

Abstract

The disease caused by the duck Tembusu virus (DTMUV) is one of the most prevalent arthropod-borne viral diseases in poultry. DTMUV is classified into three distinct clusters based on significant genetic divergence: Cluster 1, Cluster 2 (subdivided into 2.1 and 2.2), and Cluster 3. The virulence of DTMUV in ducks is potentially associated with the virus genotype. The evaluation of different clusters of DTMUV is based predominantly on the characterization of infected duck hosts, and limited attention has been paid to understanding viral virulence toward the infected mosquito vectors. In this study, we explore the infectivity patterns of DTMUV Cluster 1 (DTMUV 1) and Cluster 2.1 (DTMUV 2.1) in the primary mosquito vector, Culex tritaeniorhynchus. Our objective was to explore the relationship between the mosquito vector and DTMUV genotype, intending to determine whether the mosquito vector alters viral biology, thereby influencing the consequential infectivity characteristics in the host cells. We found that variation in viral nonstructural protein-5 (an RNA-dependent RNA polymerase) may influence the antigenicity process in Cx. tritaeniorhynchus. Our results revealed DTMUV1 underwent higher replication than DTMUV2.1 in mosquito salivary glands and saliva. Furthermore, DTMUV1 derived from mosquito saliva produced larger plaque sizes in baby hamster kidney-21 (BHK-21) cells than DTMUV2.1 derived from mosquito saliva. Interestingly, DTMUV2.1 was more efficient than DTMUV1 in inducing the production of mRNAs for macroglobulin complement-related factor, thioester-containing protein, and antimicrobial peptides (cecropin family) within the mosquito salivary gland. Our findings collectively suggest that Cx. tritaeniorhynchus can influence an environment conducive to modifying the amino acid composition of DTMUV1 and DTMUV2.1 in a manner that may affect the innate immune response, consequently augmenting viral virulence.

Keywords: BHK-21 cell; Culex tritaeniorhynchus; Duck Tembusu virus; Innate immune response; NS5.

Graphical abstract

Fig. 1

Phylogeny of DTMUV based on the partial NS5 gene. The tree with the highest log likelihood is shown. Evolutionary analyses were conducted in MEGA X and phylogenies were inferred using the Maximum Likelihood method and the Tamura-Nei model. The newly generated sequences are indicated in red.

Fig. 2

Multiple sequence alignment of clusters 1 and 2.1 of DTMUV (DTMUV1 and DTMUV2.1). Members of DTMUV1 include AVM38076 (reference gene), PP507054 (derived from cell culture), and PP507052 (derived from the saliva of Cx. tritaeniorhynchus. Members of DTMUV2.1 include QBA86037 (reference gene), PP507053 (derived from cell culture), and PP507050 (derived from the saliva of Cx. tritaeniorhynchus). Black highlighting indicates identical residues; red indicates similar to consensus; no highlighting indicates different from consensus.

Fig. 3

Infections of DTMUV clusters 1 and 2.1 in Cx. tritaeniorhynchus. DTMUV1 and DTMUV2.1 were detected by qRT-PCR in the mosquito body (A), salivary gland (B), and saliva (C). Total RNA levels are expressed as log₁₀ DTMUV genome copy number per 250 ng. Each bar represents an average of 30 samples, totaling 180 samples per experiment. These results are from three experiments, and the data are presented as the mean ± standard error (SEM). Statistically significant differences between clusters 1 and 2.1 were analyzed using unpaired t-tests. Asterisks (∗) indicate statistically significant differences: ∗∗∗∗P < 0.0001.

Fig. 4

Effects of DTMUV clusters 1 and 2.1 derived from Cx. tritaeniorhynchus saliva on BHK-21 cell cultures. A Different DTMUV clusters were analyzed by plaque and immunostaining assay including DTMUV1 from Cx. tritaeniorhynchus saliva-derived, DTMUV2.1 from Cx. tritaeniorhynchus saliva-derived, and Uninfected BHK-21 cells treated as control. B Variation in plaque-size of DTMUV1 and DTMUV2.1 derived from infected saliva. Compare the plaque size between clusters 1 and 2.1. C Concentrations of DTMUV1 and DTMUV2.1 from infected mosquito saliva were titrated using TCID₅₀ analysis. Each bar includes saliva from 90 samples, totaling 180 samples in this experiment. The results were performed in triplicate experiments, and the data are presented as the mean ± standard error (SEM). Statistically significant differences between groups were analyzed using an unpaired t-test. Asterisks (∗) indicate statistically significant differences: ∗P < 0.05, ∗∗∗P < 0.001.

Fig. 5

Innate immune response to DTMUV clusters 1 and 2.1 in Cx tritaeniorhynchus. Transcription levels of thioester-containing protein-3 of Culex tritaeniorhynchus (TEP3) (A), macroglobulin complement-related factor of Cx. tritaeniorhynchus (MCR) (B), and antimicrobial peptides (AMPs) within the salivary glands of Cx. tritaeniorhynchus infected with DTMUV1 and DTMUV2.1 (C). The results are from an experiment with three replications, and the data are presented as the mean ± standard error (SEM). Differences between groups were analyzed using unpaired t-tests. Asterisks (∗) indicate statistically significant differences: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

Fig. 6

Effect of innate immune on DTMUV1 and DTMUV2.1 within the salivary glands of Cx. tritaeniorhynchus.A-B Transcription levels of DTMUV1 (A) and CEC A, B, and C (B) of the DTMUV1- infected mosquitoes. C-D Transcription levels of DTMUV2.1 (C) and CEC A, B, and C (D) of the DTMUV2.1-infected mosquitoes. The data are presented as the mean ± standard error (SEM). Differences between groups were analyzed using unpaired t-tests. Asterisks (∗) indicate statistically significant differences: ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Abbreviation: ns, non-significant.