Genetic characterization of a novel picorna-like virus in Culex spp. mosquitoes from Mozambique

Abstract

Background: Mosquitoes are the potential vectors for a variety of viruses that can cause diseases in the human and animal populations. Viruses in the order Picornavirales infect a broad range of hosts, including mosquitoes. In this study, we aimed to characterize a novel picorna-like virus from the Culex spp. of mosquitoes from the Zambezi Valley of Mozambique.

Methods: The extracted RNA from mosquito pools was pre-amplified with the sequence independent single primer amplification (SISPA) method and subjected to high-throughput sequencing using the Ion Torrent platform. Reads that are classified as Iflaviridae, Picornaviridae and Dicistroviridae were assembled by CodonCode Aligner and SPAdes. Gaps between the viral contigs were sequenced by PCR. The genomic ends were analyzed by 5' and 3' RACE PCRs. The ORF was predicted with the NCBI ORF finder. The conserved domains were identified with ClustalW multiple sequence alignment, and a phylogenetic tree was built with MEGA. The presence of the virus in individual mosquito pools was detected by RT-PCR assay.

Results: A near full-length viral genome (9740 nt) was obtained in Culex mosquitoes that encoded a complete ORF (3112 aa), named Culex picorna-like virus (CuPV-1). The predicted ORF had 38% similarity to the Hubei picorna-like virus 35. The sequence of the conserved domains, Helicase-Protease-RNA-dependent RNA polymerase, were identified by multiple sequence alignment and found to be at the 3' end, similar to iflaviruses. Phylogenetic analysis of the putative RdRP amino acid sequences indicated that the virus clustered with members of the Iflaviridae family. CuPV-1 was detected in both Culex and Mansonia individual pools with low infection rates.

Conclusions: The study reported a highly divergent, near full-length picorna-like virus genome from Culex spp. mosquitoes from Mozambique. The discovery and characterization of novel viruses in mosquitoes is an initial step, which will provide insights into mosquito-virus interaction mechanisms, genetic diversity and evolution.

Keywords: Culex; Iflavirus; Mosquitoes; Picorna-like virus; RNA virus.

Fig. 1

Schematic presentation of the genome of CuPV-1. The HTS reads were mapped back to the sequenced genome using Bowtie2 to display the coverage and sequence depth (upper panel). The ORF corresponds to the entire open box (lower panel). The numbers above the ORF indicate the nucleotide positions and below are the amino acid positions. In the box, the position of the putative structural proteins (1A to D) and the non-structural proteins (Leader peptide, L; Helicase; Protease; RNA-dependent RNA polymerase, RdRP) are shown. The dark areas in the ORF represent regions containing conserved sequences in the viral structural or the non-structural proteins. The dotted-arrow represents the identified cleavage site. The approximate positions of the structural and non-structural proteins were identified by the sequence similarity of other picorna-like viruses

Fig. 2

Comparison of the deduced amino acid sequences of the non-structural proteins of CuPV-1 and 11 other picorna-like viruses. a Alignment of the conserved regions of the putative RNA helicase region. (Full names and references of these viruses are shown in Table 1). The motifs identified by Koonin and Dolja (1993) are labeled A, B, and C. b Alignment of the putative protease domain of CuPV-1 with those of other viruses. c Alignment of the putative RNA-dependent RNA polymerase domain of CuPV-1 with those of other viruses, which are labeled I-VIII. The conserved residues are marked with asterisks (*) and residues that are identical in more than 60% of the sequences are shown in dark shades

Fig. 3

Phylogenetic analysis of the putative RdRP region. The Maximum Likelihood tree was produced, and bootstrapped at 500 replicates using MEGA7 software and involved 37 amino acid sequences, including CuPV-1. Viruses and references are listed in Table 1