Multiple Genome Sequences of Foot-and-Mouth Disease Virus Asia-1 Lineage Sindh-08 from Outbreaks in Pakistan, 2011 to 2012.

We report the near-full-length genome sequences of 22 isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1, lineage Sindh-08, obtained from foot-and-mouth disease outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these new genomes for understanding the regional molecular epidemiology.

ANNOUNCEMENT

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV; genus Aphthovirus, family Picornaviridae) is a viral disease of livestock of high socioeconomic importance (1–3). FMDV exists as seven serotypes with multiple lineages and subtypes that are clinically indistinguishable (4). Continuous genomic surveillance of the FMDVs circulating in regions of endemicity is therefore critical to inform and update preventative vaccination strategies (5, 6).

The viruses reported herein (n = 22) were isolated from epithelial samples obtained from cattle and Asian buffalo (Bubalus bubalis) with clinical signs consistent with FMD (Table 1): vesicular lesions on the feet and in the mouth, combined with various degrees of lameness and hypersalivation. The samples were collected as part of FMD surveillance carried out by government officials in the Federal Islamabad Capital Territory, Azad Jammu and Kashmir Administrative Region, and three provinces, Punjab, Sindh, and Khyber Pakhtunkhwa (7). There were no institutional approvals required for this work.

TABLE 1

Sampling locations, sequencing metrics, and accession numbers for the sequences herein

Isolate	Sampling location	Host species	Genome length (nt)	No. of mapped reads	Avg coverage (no. of reads)	Avg read length (nt)	GC content (%)	GenBank accession no.	SRA accession no.
Asia1/PAK/FSD/85/2012	Faisalabad	Cattle	8,081	615,741	11,159	149	54	OM471659	SAMN25813628
Asia1/PAK/ICT/271/2012	Islamabad	Buffalo	8,085	955,754	17,322	149	54	OM471660	SAMN25813629
Asia1/PAK/JHG/5/2012	Jhang	Cattle	8,075	657,137	11,854	148	54	OM471661	SAMN25813630
Asia1/PAK/KCH/16/2012	Karachi	Cattle	8,071	220,056	4,001	150	54	OM471662	SAMN25813631
Asia1/PAK/KCH/17/2012	Karachi	Cattle	8,084	442,956	8,066	150	54	OM471663	SAMN25813632
Asia1/PAK/KCH/32/2011	Karachi	Buffalo	8,071	352,595	6,403	149	54	OM471664	SAMN25813633
Asia1/PAK/KCH/34/2011	Karachi	Buffalo	8,082	1,476,764	26,747	149	54	OM471665	SAMN25813634
Asia1/PAK/KCH/38/2011	Karachi	Buffalo	8,081	1,637,807	29,617	149	54	OM471666	SAMN25813635
Asia1/PAK/KCH/39/2011	Karachi	Buffalo	8,081	1,350,356	24,502	149	54	OM471667	SAMN25813636
Asia1/PAK/KHT/9/2012	Kohat	Cattle	8,072	329,045	5,983	150	54	OM471668	SAMN25813637
Asia1/PAK/MRD/66/2012	Mardan	Cattle	8,071	104,177	1,889	149	54	OM471669	SAMN25813638
Asia1/PAK/RWP/227/2012	Rawalpindi	Buffalo	8,081	347,267	6,315	150	54	OM471670	SAMN25813639
Asia1/PAK/RYK/280/2012	Rahim Yar Khan	Cattle	8,078	340,211	6,169	149	55	OM471671	SAMN25813640
Asia1/PAK/RYK/283/2012	Rahim Yar Khan	Cattle	8,083	1,277,708	23,103	149	55	OM471672	SAMN25813641
Asia1/PAK/RYK/287/2012	Rahim Yar Khan	Cattle	8,071	254,331	4,602	149	55	OM471673	SAMN25813642
Asia1/PAK/SGD/22/2011	Sargodha	Cattle	8,074	368,016	6,680	149	54	OM471674	SAMN25813643
Asia1/PAK/SGD/23/2011	Sargodha	Cattle	8,071	443,911	8,069	150	54	OM471675	SAMN25813644
Asia1/PAK/SGD/25/2011	Sargodha	Cattle	8,075	417,625	7,597	150	54	OM471676	SAMN25813645
Asia1/PAK/SGD/167/2012	Sargodha	Buffalo	8,088	665,120	12,095	150	54	OM471677	SAMN25813646
Asia1/PAK/SGD/171/2012	Sargodha	Cattle	8,079	235,473	4,274	150	55	OM471678	SAMN25813647
Asia1/PAK/SGD/170/2012	Sargodha	Cattle	8,078	770,428	13,955	149	55	OM471679	SAMN25813648
Asia1/PAK/SGD/179/2012	Sargodha	Buffalo	8,075	214,297	3,888	149	55	OM471680	SAMN25813649

FMDV was confirmed by virus isolation (VI) on LFBKαvβ6 cells, followed by the detection of viral RNA in the VI supernatant by reverse transcription-quantitative PCR (qRT-PCR) (8, 9). This was achieved by infecting T25 flasks with LFBKαvβ6 cell monolayers with the samples and harvesting the supernatant by subjecting the flasks to one freeze-thaw cycle once full cytopathic effect was observed (at 24 to 72 h post-infection). The VI supernatant RNA was subjected to viral deep sequencing as previously described (10). Briefly, RNA was extracted using the MagMAX total RNA isolation kit, and the host DNA was depleted using the DNA-free DNase kit (Ambion). Samples were reverse transcribed using the Superscript II first-strand synthesis system (Invitrogen), coupled with random primers and two FMDV-specific primers (10). Double-stranded cDNA (ds-cDNA) was generated using the NEBNext Ultra II nondirectional RNA second-strand synthesis module (New England BioLabs) and purified using SPRIselect beads (Beckman Coulter). A sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina) and sequenced on the NextSeq 550 platform with the 300-cycle kit (2 × 150-bp paired-end format). All analyses were performed using CLC Genomics Workbench v21.0 with default parameters, with the exception of the following: match score, 3; mismatch penalty, 3; length fraction, 0.8; and ignore nonspecific matches. The paired-end reads were quality trimmed, then de novo assembled, and mapped to a previously published Asia-1 isolate (GenBank accession no. KM268898), representative of strains circulating in the region. All de novo assemblies were identical to the mapped assemblies in their covered regions; however, the mapped assemblies covered more of the genome. A consensus sequence was extracted from each mapping (Table 1). Annotations were copied from the reference to each consensus sequence. The 8,071 to 8,088-nucleotide (nt) genomes encode a 6,990-nt open reading frame (ORF), flanked by a 1,080 to 1,094-nt 5′ untranslated region (UTR) and a 90 to 92-nt 3′ UTR, excluding the poly(A) tail. The pairwise identity among these sequences was 94.7% to 99.9%. A BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search using the nucleotide collection database showed that the sequences shared 94.1% to 98.0% identity to the Asia-1 isolate TUR/13/2013 (KM268898), which was isolated from a cow in the Gündoğan region of Turkey in 2013 (11). These viruses were previously determined to belong to the Asia-1/Sindh-08 lineage based on the VP1 coding region (7). These findings provide important insights into the genomic diversity and evolution within the Asia-1 serotype of FMDV circulating in Pakistan (12).

Data availability.

The genome nucleotide sequences have been deposited at GenBank under accession no. OM471659 to OM471680. The raw sequence data are available at the NCBI Sequence Read Archive under BioProject accession no. PRJNA804891. Hyperlinks to the Sequence Read Archive (SRA) are included in Table 1.