Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico

Abstract

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

Fig. 1.

Coding capacity of MERDV sequence. Main ORFs, as well as minor ORFs unlikely to be expressed, are indicated along a scaled representation of the anti-genomic strand. The size of minor ORFs is indicated by their codon number: blue, frame 1; green, frame 2; red, frame 3.

Fig. 2.

Phylogenetic tree for MERDV and selected other rhabdovirus sequences. L protein amino acid sequences were aligned using muscle (Edgar, 2004). A maximum-likelihood phylogenetic tree was estimated using the Bayesian Markov chain Monte Carlo method implemented in MrBayes version 3.2.3 (Ronquist et al., 2012) sampling across the default set of fixed amino acid rate matrices with 10 million generations, discarding the first 25 % as burn-in. The original figure was produced using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The tree is midpoint-rooted and selected nodes are labelled with posterior probability values. Rhabdovirus genera, where defined, are labelled on the far right. GenBank accession numbers are indicated next to virus names. Bar indicates amino acid substitutions per site.

Fig. 3.

Relative UpA and CpG frequencies in the L protein ORF of different rhabdovirus species. UpA and CpG frequencies were calculated in two different ways. (a) In each sequence, the numbers of UpA and CpG dinucleotides, and A, C, G and U mononucleotides, were counted. Dinucleotide frequencies, fXpY, were expressed relative to their expected frequencies, fX × fY, in the absence of selection. (b) To factor-out codon and amino acid usage, 1000 shuffled ORF sequences were generated for each virus sequence. In each shuffled sequence, the original amino acid sequence and the original total numbers of each of the 61 codons were maintained, but synonymous codons were randomly shuffled between the different sites where the corresponding amino acid is used in the original sequence. Next, the UpA and CpG frequencies in the original sequence were expressed relative to their mean frequencies in the codon-shuffled sequences. As codon usage is factored-out, the UpA and CpG relative frequencies tend to be less extreme in (b) compared with (a). Each point represents a single rhabdovirus sequence. Solid points correspond to species within defined genera, colour coded by genus (see key). Annotated open circles correspond to species that are currently unassigned at genus level, colour coded by host (or presumed host) taxa. Asterisks in the key indicate clades with uncertain host taxa: viruses in the unclassified ‘arthropod-infecting’ clades (yellow open circles) were isolated from arthropods but not from vertebrates; the sole representative of the genus Tupavirus was isolated from mammals but not from arthropods, although its phylogenetic position suggests that it may be arthropod-borne; the presence of viruses derived from vertebrates and viruses derived from arthropods in each of the unclassified ‘vertebrate (arthropod-borne)’ clades (brick-red open circles) suggests that all of these viruses are likely arboviruses. GenBank accession numbers of sequences used: NC_000855, NC_000903, NC_001542, NC_001560, NC_001615, NC_001652, NC_002251, NC_002526, NC_002803, NC_003243, NC_003746, NC_005093, NC_005974, NC_005975, NC_006429, NC_006942, NC_007020, NC_007642, NC_008514, NC_009527, NC_009528, NC_011532, NC_011542, NC_011639, NC_013135, NC_013955, NC_016136, NC_017685, NC_017714, NC_018381, NC_018629, NC_020803, NC_020804, NC_020805, NC_020806, NC_020807, NC_020808, NC_020809, NC_020810, NC_022580, NC_022581, NC_022755, NC_024473, NC_025251, NC_025253, NC_025255, NC_025340, NC_025341, NC_025342, NC_025353, NC_025354, NC_025356, NC_025358, NC_025359, NC_025362, NC_025364, NC_025365, NC_025371, NC_025376, NC_025377, NC_025378, NC_025382, NC_025384, NC_025385, NC_025387, NC_025389, NC_025391, NC_025392, NC_025393, NC_025394, NC_025395, NC_025396, NC_025397, NC_025398, NC_025399, NC_025400, NC_025401, NC_025405, NC_025406, NC_025408.