ABSTRACT
In this report, we describe eight complete genome sequences of African horse sickness virus (AHSV) strains belonging to four different serotypes, namely, AHSV-5, AHSV-6, AHSV-8, and AHSV-9. Samples were collected in Namibia and South Africa from infected horses between 2000 and 2011. As expected, phylogenetic analyses of the variable outer capsid protein VP2 genomic sequences of AHSV-6 and AHSV-8 show higher nucleotide identity between the isolated viruses than that of the relevant reference strains. The full-genome sequence of AHSV will provide useful information on its geographical origin, and it will also be instrumental for comparing the distribution of the Namibian isolate with that of global isolates.
ANNOUNCEMENT
African horse sickness (AHS) is a major arthropod-borne disease affecting all Equidae species and is caused by the African horse sickness virus (AHSV) belonging to the Orbivirus genus within the Sedoreoviridae family (1–3). AHSV is a nonenveloped virus structured into two concentric protein shells surrounding the genome of 10 double-stranded RNA (dsRNA) linear segments designated from segment 1 (S1) to S10 carrying 7 structural proteins (VP1 to VP7) and 5 nonstructural proteins (NS1 to NS4 and NS3A) (3–6).
In this work, blood samples were collected from animals suspected of infection during the febrile stage of disease or from infected organs of dead animals at necropsy (Table 1). Virus isolation was performed on Kenyon Culicoides cell (KC) lines and was subsequently amplified on BSR cells (a clone of baby hamster kidney-21 cells) (7, 8). Viral dsRNA was extracted as described previously from cell lysates (9). Full-length amplification of cDNAs was performed with the full-length amplification of cDNA (FLAC) method followed by reverse transcription-PCR (RT-PCR) (10). Amplified DNA was subsequently purified using DNA clean and concentrator 5 kit according to the manufacturer’s instructions (Zymo Research) and was quantified with the Qubit 2.0 fluorometer (Thermo Fisher Scientific).
TABLE 1.
Description of eight AHSV genome sequences obtained from samples collected from infected horses in Namibia and South Africa
| Virus protein | Data by virus name |
|||||
|---|---|---|---|---|---|---|
| AHSV-5 Swanepoel South Africa (lung)a |
AHSV-6 Okahandja Namibia (spleen)b |
|||||
| No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | |
| VP1 | 125,286 (10.91%) | 6,581 | OP455575 | 87,499 (11.12) | 5,660 | OP455585 |
| VP2 | 267,479 (23.29) | 7,244 | OP455576 | 178,028 (22.63) | 7,131 | OP455586 |
| VP3 | 196,754 (17.13) | 7,067 | OP455577 | 103,004 (13.09) | 6,544 | OP455587 |
| VP4 | 20,041 (1.75) | 2,930 | OP455578 | 66,190 (8.41) | 6,520 | OP455588 |
| NS1 | 57,759 (5.03) | 6,890 | OP455579 | 28,460 (3.62) | 4,791 | OP455589 |
| VP5 | 99,598 (8.67) | 6,614 | OP455580 | 76,416 (9.71) | 6,758 | OP455590 |
| VP7 | 42,505 (3.70) | 6,117 | OP455581 | 28,473 (3.62) | 4,873 | OP455591 |
| NS2 | 112,004 (9.75) | 6,387 | OP455582 | 15,838 (2.01) | 3,964 | OP455592 |
| VP6 | 21,479 (1.87) | 4,657 | OP455583 | 39,599 (5.03) | 5,948 | OP455593 |
| NS3 | 32,005 (2.79) | 5,647 | OP455584 | 10,465 (1.33) | 3,378 | OP455594 |
| AHSV-6 Omaruru Namibia (blood)c |
AHSV-9 Okahandja Namibia (blood)d |
|||||
| No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | |
| VP1 | 6,746 (0.46) | 560 | OP508188 | 5,847 (0.46) | 481 | OP508198 |
| VP2 | 12,376 (0.85) | 1,252 | OP508189 | 3,809 (0.30) | 416 | OP508199 |
| VP3 | 39,382 (2.70) | 4,555 | OP508190 | 17,905 (1.42) | 2,081 | OP508200 |
| VP4 | 45,338 (3.11) | 5,702 | OP508191 | 34,884 (2.76) | 5,191 | OP508201 |
| NS1 | 251,453 (17.26) | 7,584 | OP508192 | 94,529 (7.49) | 7,284 | OP508202 |
| VP5 | 124,283 (8.53) | 7,535 | OP508193 | 41,519 (3.29) | 6,385 | OP508203 |
| VP7 | 9,902 (0.68) | 2,705 | OP508194 | 194,471 (15.41) | 7,404 | OP508204 |
| NS2 | 138,409 (9.50) | 7,362 | OP508195 | 174,371 (13.81) | 7,383 | OP508205 |
| VP6 | 282,733 (19.41) | 7,486 | OP508196 | 14,653 (1.16) | 3,868 | OP508206 |
| NS3 | 155,235 (10.66) | 7,670 | OP508197 | 69,841 (5.53) | 4,833 | OP508207 |
| AHSV-8 Windhoek/A Namibia (lung)e |
AHSV-8 Windhoek/B Namibia (lung)f |
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| No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | |
| VP1 | 62,747 (13.11) | 4,924 | OP432769 | 115,763 (8.81) | 6,440 | OP455595 |
| VP2 | 86,772 (18.13) | 6,606 | OP432770 | 119,618 (9.10) | 7,077 | OP455596 |
| VP3 | 59,395 (12.41) | 5,244 | OP432771 | 230,920 (17.57) | 7,246 | OP455597 |
| VP4 | 26,266 (5.49) | 4,170 | OP432772 | 46,609 (3.55) | 5,894 | OP455598 |
| NS1 | 35,886 (7.50) | 5,509 | OP432773 | 86,966 (6.62) | 7,295 | OP455599 |
| VP5 | 35,661 (7.45) | 6,026 | OP432774 | 77,256 (5.88) | 6,905 | OP455600 |
| VP7 | 15,852 (3.31) | 4,292 | OP432775 | 97,321 (7.40) | 7,214 | OP455601 |
| NS2 | 32,426 (6.78) | 5,843 | OP432776 | 140,240 (10.67) | 7,209 | OP455602 |
| VP6 | 3,932 (0.82) | 1,072 | OP432777 | 16,559 (1.26) | 4,003 | OP455603 |
| NS3 | 7,621 (1.59) | 3,731 | OP432778 | 18,616 (1.42) | 5,125 | OP455604 |
| AHSV-6 Gobabis Namibia (liver)g |
AHSV-8 Windhoek/C Namibia (spleen)h |
|||||
| No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | No. of mapped reads (%) | Final coverage (×) | GenBank accession no. | |
| VP1 | 7,829 (4.15) | 624 | OP508208 | 35,274 (12.04) | 2,787 | OP455605 |
| VP2 | 17,533 (9.30) | 1,723 | OP508209 | 29,628 (10.11) | 2,878 | OP455606 |
| VP3 | 18,603 (9.87) | 2,106 | OP508210 | 24,507 (8.36) | 2,764 | OP455607 |
| VP4 | 5,320 (2.82) | 851 | OP508211 | 13,954 (4.76) | 2,237 | OP455608 |
| NS1 | 8,545 (4.53) | 1,600 | OP508212 | 27,355 (9.34) | 4,451 | OP455609 |
| VP5 | 9,286 (4.93) | 1,871 | OP508213 | 12,260 (4.18) | 2,469 | OP455610 |
| VP7 | 2,114 (1.12) | 573 | OP508214 | 7,070 (2.41) | 1,929 | OP455611 |
| NS2 | 6,984 (3.70) | 1,885 | OP508215 | 17,700 (6.04) | 4,739 | OP455612 |
| VP6 | 18,809 (9.98) | 5,152 | OP508216 | 646 (0.22) | 198 | OP455613 |
| NS3 | 4,402 (2.33) | 2,150 | OP508217 | 11,510 (3.93) | 4,860 | OP455614 |
Libraries were prepared using Nextera XT library prep kit (Illumina Inc., San Diego, CA) according to the manufacturer’s protocol. Deep sequencing was performed on the NextSeq500 instrument (Illumina Inc.) using the NextSeq 500/550 mid output reagent cartridge v2 (300 cycle) (Illumina Inc.) and standard 150-bp paired-end reads.
Read quality control was performed using fastqc v0.11.5, and the reads were trimmed using trimmomatic v0.36 (11) using the following parameters: ILLUMINACLIP:/trimmomatic-0.36-6/adapters/NexteraPE-PE.fa:2:30:10 LEADING:25 TRAILING:25 SLIDINGWINDOW:20:25 MINLEN:36. The resulting high-quality reads underwent a depletion step to remove the host reads using Bowtie 2.1.0 and the GCF_000349665 Mesocricetus host genome as a reference (12). The remaining reads were de novo assembled using SPAdes assembler v3.11 (13). In order to identify the best reference sequences, the scaffolds were compared to the nucleotide and nonredundant databases using BLAST (14). Full genomes were obtained by mapping the reads to the identified AHSV reference sequences using Bowtie 2.1.0 (12) and by subsequently using the MegAlign software (v17.2, DNASTAR, Inc., Madison, WI) (15). Sequences were submitted to NCBI and accession numbers within the reference used, the mapped reads, and the coverage obtained are detailed in Table 1 (16). All software tools were run with default parameters unless otherwise specified.
The phylogenetic tree of VP2 gene nucleotide sequences (segment 2) was elaborated using the maximum likelihood RAxML method and was constructed using full-length genomic sequences in a pair-wise deletion, p-distance algorithm and was bootstrapped using 100 replicates using MegAlign software. The different VP2 genes identifying the different serotypes are indicated in Fig. 1 together with outgroups.
FIG 1.
Phylogenetic trees of AHSV genome segment 2. VP2 genomic sequences from the 7 Namibian and 1 South African virus isolates together with the AHSV reference strains were used to generate the phylogenetic tree using maximum likelihood RAxML and bootstrapped values were obtained using 100 replicates. VP2 sequences of this study are in bold. Newly isolated viral sequences of AHSV-6 and AHSV-8 show higher nucleotide identity between circulating serotypes than the relevant reference virus strains.
In conclusion, in this work, we describe the sequence analysis of eight AHSVs representing four different serotypes (AHSV-5, AHSV-6, AHSV-8, and AHSV-9), isolated from infected horses during AHS outbreaks which occurred in Namibia and South Africa between 2000 and 2011 (Table 1). The data presented increases the low number of full-length AHSV genomes publicly available and provide useful information on the geographical origin of the circulating AHSV strains.
Data availability.
The complete genome sequences of the eight AHSV strains are available in GenBank, under accession numbers listed in Table 1. The reference genomes used for the annotation were published previously (16). AHSV sequences raw data were submitted to SRA under BioProject PRJNA930321.
ACKNOWLEDGMENT
This study was funded by the Italian Ministry of Health with grant no. IZS AM 01.17RC.
Contributor Information
Marco Caporale, Email: m.caporale@izs.it.
Kenneth M. Stedman, Portland State University
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The complete genome sequences of the eight AHSV strains are available in GenBank, under accession numbers listed in Table 1. The reference genomes used for the annotation were published previously (16). AHSV sequences raw data were submitted to SRA under BioProject PRJNA930321.