Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach

Abstract

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies.

Fig. 1.

Linear representation of the GpSGHV-Eth genome: The linearization is presented starting with the ATG initiating codon of p74 (SGHV-Eth001). The arrows indicate the positions and orientations of the transcription potential of each ORF. In total, 112 ORFs in the GpSGHV-Eth genome were identical in length (amino acid residues) to the homologous ORFs in the GpSGHV-Uga genome; 37 ORFs had insertions and/or deletions, 11 ORFs were deleted and 24 ORFs were novel in the GpSGHV-Eth genome compared with the GpSGHV-Uga genome. The ORFs are colour-coded to show their homology to the corresponding ORFs in the GpSGHV-Uga genome. The abbreviations used are explained in the footnote to Table 1. The figure is drawn to scale.

Fig. 2.

Genome synteny visualization of the relationship between GpSGHV-Eth and GpSGHV-Uga. The positions of the genomes are shown between the thick black lines, which represent the dsDNA viral genomes. (a) The dot-plot was generated from the whole-genome DNA homology alignments between GpSGHV-Eth and GpSGHV-Uga genomic sequences. The red and blue colours indicate the sequence direction (reverse and forward). (b, c) Syntenic maps show the overall collinearity of GpSGHV-Eth and GpSGHV-Uga compared with Musca domestica salivary gland hypertrophy virus (MdSGHV). The red lines (b, c) indicate identity levels between the viruses, whilst the blue lines (c) indicate instances of inversions. The bands represented in the thick black lines do not necessarily indicate the ORFs, but rather the conserved genomic regions.

Fig. 3.

Repeat status and genetic heterogeneity in the GpSGHV-Eth genome. (a) Approximately 30 and 70 % of the 89 508–89 648 nt region were covered by short repeats of 30 and 26 nt, respectively. (b) Two repeat sequences, 27 nt each, occur two and three times in 65 and 35 %, respectively, of the reads in the 183 153–183 354 nt region.

Fig. 4.

Loci of the GpSGHV-Eth genome with repeating elements. (a) The arrows represent the repeat core elements in the indicated directions. The name, type, size, copy number and genomic location of the repeat are indicated on the right. DR, direct repeat; IR, indirect repeat. Parameters: length >50 nt, n>2. (b) Alignment of the consensus nucleotide sequences of the direct repeats R9, R10, R11, R12, R13 and R14.

Fig. 5.

Inserted and deleted nucleotides in the GpSGHV-Eth genome compared with the GpSGHV-Uga genome. Positive and negative numbers indicate insertions and deletions, respectively, in the two virus genomes.