Complete Genome Sequence of a Severe Acute Respiratory Syndrome-Related Coronavirus from Kenyan Bats.

We identified a strain of betacoronavirus BtKY72/Rhinolophus sp./Kenya/2007 (here BtKY72) from rectal swab samples in Kenyan bats. This paper reports the complete genomic sequence of BtKY72, which is closely related to BtCoV/BM48-31/Bulgaria/2008, a severe acute respiratory syndrome (SARS)-related virus from Rhinolophus bats in Europe.

ANNOUNCEMENT

The 2002 and 2003 outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) infection was a significant public health threat at the beginning of the 21st century (1–6). Initial identification of SARS-CoV in civet cats and other wild animals in live animal markets suggests zoonosis (7). Later, Rhinolophus sp. bats were identified as harboring severe acute respiratory syndrome-related CoV at high frequencies and were believed to be a natural reservoir host for SARS-CoV (8, 9).

During a 5-year bat coronavirus (CoV) surveillance study (2006 to 2010) in Kenya, we identified five bat betacoronaviruses by pan-CoV reverse transcription-PCR (RT-PCR) from fecal samples of Chaerephon and Rhinolophus bats (10, 11). The Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and Kenya Wildlife Services approved all protocols related to the animal experiments in this study. These bat betacoronaviruses shared >98% nucleotide identity with each other and were clustered with other known bat SARS-related CoVs identified from Rhinolophus bats in China and Europe (8, 9, 12–15) based on a short amplicon sequence of open reading frame 1b (ORF1b) (121 bp). We selected RNA from the BtKY72 bat, which was one of the five betacoronavirus-positive bats from a previous study (11), for full genome sequencing. To determine the full genome sequence, consensus degenerate primers were designed from conserved sequences based on all known SARS-related CoVs (Table 1). Several small islands of sequences scattered throughout the genome were first determined from a Kenyan Rhinolophus bat using sets of seminested or nested consensus RT-PCR primers by Sanger sequencing. Then, sets of sequence-specific primers were used to fill the gaps and generate the full genome sequence, named BtKY72/Rhinolophus sp./Kenya/2007 (Table 1). The 5′ and 3′ ends of genome sequences were determined using a 5′/3′ rapid amplification of cDNA ends (RACE) kit (Roche). Complete genome sequencing was not performed due to limited viral loads in fecal samples from the other four betacoronavirus-positive bats.

TABLE 1

Genomic PCR primers used in this study

PCR or primer no.	First-round PCR primer			Nested-round PCR primer
	Name	Sequence (5′→3′)	Nucleotide positiona	Name	Sequence (5′→3′)	Nucleotide positiona
Consensus degenerate PCR primers
1	F20_Fwd	TACCCAGGAAAAGCCAACCAACC	15–37	F20_Fwd	TACCCAGGAAAAGCCAACCAACC	15–37
	R328new_Rev	TGTAAAACAGGTAAACTGAGTTGGACGTG	296–324	R300_Rev	TGAAACCAGGGACAAGGCTCTCC	254–284
2	F180_Fwd	AGACTGCAGACTGCTTACGGTTTCG	174–198	F220_Fwd	CATCAGCATACCTAGGTTTCGTCCG	216–240
	R700_Rev	CACCTAACTCATAAGACTTTAGATCGATGCC	668–698	R490_Rev	CATCAGATCGTTTAATGAACACATAGGGC	457–485
3	F1440_Fwd	ATTGAAACTCGACTCCGCAAGGG	1436–1458	F1470_Fwd	GGTAGGACTARATGTTTTGGRGGYTGTG	1460–1487
	R2090_Rev	TACAAGACCACCWGTIACATAYGCCATRA	2050–2079	R2090_Rev	TACAAGACCACCWGTIACATAYGCCATRA	2050–2079
4	F5810_Fwd	CAGAATATAAAGGACCAGTGACTGATGTTTTC	5691–5722	F5810_Fwd	CAGAATATAAAGGACCAGTGACTGATGTTTTC	5691–5722
	R6580_Rev	GCTCGTTAGGTTTCTTAATGGTAATGCTTG	6429–6458	R6580_Rev	GCTCGTTAGGTTTCTTAATGGTAATGCTTG	6429–6458
5	F8330_Fwd	ATGCCCAAGTAGCAARAAGYCACAATG	8220–8246	F8330_Fwd	ATGCCCAAGTAGCAARAAGYCACAATG	8220–8246
	R9580_Rev	TGGTGAAATAGAATGTCAAGTACAAGTAAAAGA	9441–9473	R9470_Rev	TAGCAGCAACTACATGGTTGTACTCACC	9345–9372
6	F10290_Fwd	GGCTTAAAGTTGATACYTCTAAYCCTAAGACACC	10183–10216	F10290_Fwd	GGCTTAAAGTTGATACYTCTAAYCCTAAGACACC	10183–10216
	R11440_Rev	GCCCACATGGAAATAGCTTGATCTAARG	11308–11335	R11480_Rev	AACGACACCAGAATAGTTAGAGGTTACAGAA	11345–11375
7	F11190_Fwd	TCTACATGCCTGCTAGYTGGGTGATG	11079–11104	F11220_Fwd	CGTATTATGACATGGCTYGAATTGGC	11105–11130
	R12390_Rev	CGTGCATTGTTGATAATGTTGTTAAGTGC	12252–12280	R12390_Rev	CGTGCATTGTTGATAATGTTGTTAAGTGC	12252–12280
8	F15280_Fwd	ACAGGRCTATGCCTAACATGCTTAG	15170–15198	F15300_Fwd	ATTATGGCTTCTCTTGTCCTTGCTCG	15200–15225
	R15980_Rev	TTTCAATCATRAGTGTACCATCTGTTTTGAC	15849–15879	R15980_Rev	TTTCAATCATRAGTGTACCATCTGTTTTGAC	15849–15879
9	F15830_Fwd	GACCTCAYGAATTTTGCTCWCAGC	15729–15752	F15850_Fwd	TCTCAGCAYACRAATGCTAGTTAAACAAGG	15746–15775
	R16850_Rev	GTAGTACCTCTGTACACAACAGCATCWCC	16718–16746	R16840_Rev	GTACACAACAGCATCACCATAGTCACC	16709–16735
10	F16455_Fwd	TTGTGTGCTAATGGTCAGGTTTTTGG	16347–16372	F16455_Fwd	TTGTGTGCTAATGGTCAGGTTTTTGG	16347–16372
	R17560_Rev	GTGTCRACAATTTCRGCAGGACAACG	17427–17452	R17510_Rev	ATGTCWGGACCTATTGTTTTCATRAGTCTGC	17377–17407
11	F17990_Fwd	CGMAATGTGGCTACKTTACARGCAGAA	17874–17903	F17990_Fwd	CGMAATGTGGCTACKTTACARGCAGAA	17874–17903
	R19170_Rev	TTACAATTCCAAAACAARCARACACCATC	19038–19066	R19195_Rev	CATTGGCYGGRTAACGATCAACG	19069–19091
12	F18870_Fwd	CGCGTTGATTGGTCTGTTGAATAYC	18768–18792	F18870_Fwd	CGCGTTGATTGGTCTGTTGAATAYC	18768–18792
	R20100_Rev	ATGTGACTCCATTGACRCTWGCTTG	19959–19983	R20110_Rev	TTTTACTGATTCTCCAATTAATGTGACTCC	19974–20004
13	F19880_Fwd	TTTCTACAATAGGTRTCTGYACAATGACTG	19773–19802	F19900_Fwd	TGACTGACATTGMCAAGAAACCTACTG	19797–19823
	R20730_Rev	GCGTTTCACCATAATTCTGAAGGTC	20600–20625	R20730_Rev	GCGTTTCACCATAATTCTGAAGGTC	20600–20625
14	F20580_Fwd	GGTGTAAGGATGGACATGYTGAAACC	20479–20504	F20580_Fwd	GGTGTAAGGATGGACATGYTGAAACC	20479–20504
	R21200_Rev	CCACCATGAGAAATRKCCCATAAGC	21070–21096	R21210_Rev	TTGTAACAAARGCTGTCCACCATGAG	21083–21107
15	F24200_Fwd	TGGCATATAGGTTYAATGGCATTGGAG	24089–24033	F24220_Fwd	GGCATTGGAGTTRCYCAAAATGTTCTC	24109–24126
	R25345_Rev	CTCATAACAAATCCATTAAGTTCGTTTATGTG	25197–25229	R25345_Rev	CTCATAACAAATCCATTAAGTTCGTTTATGTG	25197–25229
16	F24970_Fwd	CAAAAATCATACATCACCWGATGTTGATC	24854–24882	F25005_Fwd	TTTCAGGCATTAAYGCTTCWGTCG	24894–24918
	R26290_Rev	CGCAGTAAGGATGGCTAGTGTGACTA	26127–26152	R26235_Rev	AAAGAAGTACGCTATTAACTATTAACGTACCTG	26070–26102
17	F26065_Fwd	ACACAATCGACGGCTCTTCAGGAG	25945–25968	F26120_Fwd	TGAGCCGACGACGACTACTAGCGT	25988–26011
	R26890_Rev	GATCACAGCNCCAATGACAAGTTCAC	26726–26751	R26870_Rev	CAAGTTCACTTTCCARGAGCGGTCTG	26709–26734
Specific PCR primers
1	contig10F1_Fwd	GGTAAGATGGAGAGCCTTGTCCCTG	254–278	contig10F2_Fwd	AACGAGAAAACTCACGTCCAACTCAG	284–309
	contig10R1_Rev	CTGACATAGAAGCAAGAATAATTACTACTTCCTC	1670–1703	contig10R1_Rev	CTGACATAGAAGCAAGAATAATTACTACTTCCTC	1670–1703
2	contig9-F1_Fwd	CACAAGCTGCTTGCGTGGTTAGG	1872–1894	contig9-F1_Fwd	CACAAGCTGCTTGCGTGGTTAGG	1872–1894
	contig9-R1_Rev	AGAGTTTCCATTCCTTGTGCGTCATC	6212–6237	contig9-R2_Rev	GACAACGCAAACACCACATATTGGG	6134–6158
3	contig11F1_Fwd	AGTCAAACACTTGTCTCTGAAGAAGTAGTGG	6248–6278	contig11F2_Fwd	GAAGTAGTGGAAACTCCTACCATACAGAAGG	6269–6299
	contig8-R1_Rev	GCATGATAATGTAAAACAGACTAGCAACTAATACC	8462–8495	contig8-R2_Rev	CATGTGTTATTCAATTTACCACCCTTAAGTG	8397–8427
4	contig5-F1_Fwd	TTCTACCACGTGTGTTTAGTGCTGTTG	8772–8798	contig5-F1_Fwd	TTCTACCACGTGTGTTTAGTGCTGTTG	8772–8798
	R10475_Rev	GTTAAAACCAACACTACCACATGANCCATT	10334–10363	R10410_Rev	ATTAGGTCTCATGGCACACTGRTAAACWC	10281–10309
5	Contig7-F1_Fwd	AAAATGGCAGATCAGGCTATGACCC	12129–12153	Contig7-F2_Fwd	ACAGGCTAGGTCTGAAGACAAGAGGG	12164–12189
	contig14R1_Rev	TTGTAGATTGCGGACATACTTGTCGG	15444–15469	contig14R2_Rev	CCATCAGTAGATAAGAGTGCATTCACATTAGC	15401–15432
6	500-c1-F1_Fwd	TCGATGGCCACTAATTATGACCTGAG	17229–17254	500-c1-F1_Fwd	TCGATGGCCACTAATTATGACCTGAG	17229–17254
	500-c2-R1_Rev	AGCCCAAAGGACAAACACGACTC	18369–18392	500-c2-R2_Rev	ACGCACTATGTTCCAAGGCAGACC	18442–18464
7	500-c3-F1_Fwd	AAGTTGGCATTAGGTGGTTCTGTGG	21000–21024	contig3-F2_Fwd	GCCATAAAGATTACAGAGCATTCGTGG	21024–21050
	500-R22790_Rev	CAGGTCCGATAGGTATATCACACTCATAGG	23378–23406	500-R22740_Rev	TGGCTCCTAGAAGACAACCAGCTTG	23338–23362
8	F23200_Fwd	CCGTGCTCTTTTGGTGGTGTKAGTG	23161–23185	F23200_Fwd	CCGTGCTCTTTTGGTGGTGTKAGTG	23161–23185
	500-c4-R1_Rev	CTGACATTTTAGTAGCAGCAAGATTAGCAG	24334–24361	500-c4-R2_Rev	TCTGGACTTCAGCCTCAACTTTATCAAG	24446–24475
9	500C4F1_Fwd	GCTTAGCTACTTTGTTGCATCATTCAGG	26593–26620	500C4F2_Fwd	ATTGGTGCTCATGATCATTCGTGGTT	26735–26760
	oligodT anchor_Rev	GTTTCCCAGTCACGATATTTTTTTTTTTTTTTTV	29273–29289	oligodT anchor_Rev	GTTTCCCAGTCACGATATTTTTTTTTTTTTTTTV	29273–29289

Positions relative to the genome of BtKY72/Rhinolophus sp./Kenya/2007 (GenBank accession no. KY352407).

The genome of BtKY72 was 29,259 nucleotides long, including the poly(A) tail, with 39% G+C content. Sequence alignment and a BLAST search analysis of the full-length genome sequences showed that the BtKY72 genome shared an 81% overall nucleotide identity to its nearest relative, BtCoV/BM48-3, which was identified from a Rhinolophus bat in Europe (15), and that it has 93 to 94% amino acid identity in the seven concatenated, conserved replicase domains (ADP-ribose-1″-phosphatase [ADRP], nonstructural protein 5 [nsp5], and nsp12 to nsp16) to BtCoV/BM48-31 (Fig. 1). Phylogenetic analysis suggested that BtKY72 belongs to the subgenus Sarbecovirus of the genus Betacoronavirus (Fig. 1). The genome organization contained the following gene order: 5′ UTR-ORF1ab-S-ORF3a-E-M-ORF6-ORF7a-ORF7b-N-3′ UTR. Unlike SARS-CoV and other known SARS-CoV-related bat viruses, both ORF3b and ORF8 were absent in BtKY72. ORF8 was also missing in its closest neighbor, BtCoV/BM48-31 (15).

FIG 1

Phylogenetic analysis of whole-genome sequences of betacoronaviruses. The phylogenetic tree is inferred using the maximum likelihood (ML) method available in PhyML version 3.0 (16), assuming a general time-reversible (GTR) model with a discrete gamma-distributed rate variation among sites (Γ4) and a subtree pruning and regrafting (SPR) tree-swapping algorithm. The sequences are labeled with accession number, strain name, geographic (three-letter country code), and host (species) information. BtKY72/Rhinolophus sp./Kenya/2007, sequenced in this study, is highlighted with a solid circle. The genus taxonomy information is shown to the right side of the phylogeny. The maximum likelihood bootstrap is indicated next to the nodes. The scale bar indicates the estimated number of nucleotide substitutions per site. KEN, Kenya; CHN, China; BGR, Bulgaria; NGA, Nigeria; MERS-CoV, Middle East respiratory syndrome coronavirus; HCoV, human coronavirus; MHV, mouse hepatitis virus; ZBCoV, Zaria bat coronavirus.

In conclusion, our study demonstrates that the SARS-related CoVs that were identified from Rhinolophus bats in China and Europe were also present in Kenyan Rhinolophus bats (Fig. 1). The discovery of SARS-related CoVs in Kenyan bats adds to the diversity and geographic range of CoVs in Rhinolophus bats. The genome data for BtKY72 will facilitate understanding of the molecular evolutionary characteristics of bat SARS-related CoV.

Data availability.

The complete genome sequence of BtKY72 is available in GenBank under the accession number KY352407.