Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs) have been identified in the United States that infect sheep with specific host trans- membrane protein 154 (TMEM154) diplotypes. Here, we report the first two com- plete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype A2, subgroups 1 and 2

Clawson, Michael L.

Dickey, Aaron M.

Heaton, Michael P.

Smith, Timothy P.L.

Workman, Aspen M.

English

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Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 
3-30-2017 
Complete Genome Sequences of Two Genotype A2 Small 
Ruminant Lentiviruses Isolated from Infected U.S. Sheep 
Aspen M. Workman 
USDA, ARS, U.S. Meat Animal Research Center, Clay Center, aspen.workman@ars.usda.gov 
Aaron M. Dickey 
USDA, ARS, U.S. Meat Animal Research Center, Clay Center 
Michael P. Heaton 
USDA, ARS, U.S. Meat Animal Research Center, Clay Center 
Michael L. Clawson 
USDA, ARS, U.S. Meat Animal Research Center, Clay Center 
Timothy P.L. Smith 
USDA, ARS, U.S. Meat Animal Research Center, Clay Center 
Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub 
Workman, Aspen M.; Dickey, Aaron M.; Heaton, Michael P.; Clawson, Michael L.; and Smith, Timothy P.L., 
"Complete Genome Sequences of Two Genotype A2 Small Ruminant Lentiviruses Isolated from Infected 
U.S. Sheep" (2017). Publications from USDA-ARS / UNL Faculty. 2322. 
https://digitalcommons.unl.edu/usdaarsfacpub/2322 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research 
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Complete Genome Sequences of Two
Genotype A2 Small Ruminant
Lentiviruses Isolated from Infected U.S.
Sheep
Aspen M. Workman, Aaron M. Dickey, Michael P. Heaton, Michael L. Clawson,
Timothy P. L. Smith
USDA, ARS, U.S. Meat Animal Research Center, Clay Center, Nebraska, USA
ABSTRACT Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs)
have been identified in the United States that infect sheep with specific host trans-
membrane protein 154 (TMEM154) diplotypes. Here, we report the first two com-
plete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype
A2, subgroups 1 and 2.
Small ruminant lentiviruses (SRLVs) infect sheep, goats, and wild ruminants, causingan incurable multisystemic chronic disease (1). SRLVs are phylogenetically divided
into five groups, A to E, which are further divided into different subtypes based on their
gag and pol sequences (2, 3). In the United States, genotypes A2 and B1 infect sheep
and goats (4). Viral tropism and host susceptibility to the different SRLV strains are
influenced by host and viral genotypes. In sheep, amino acid sequence variation
encoded by the transmembrane protein 154 (TMEM154) gene is associated with
susceptibility to SRLV A2 infection (5–9). On the pathogen side, two distinct SRLV A2
subgroups have evolved to infect sheep with specific TMEM154 diplotypes (8). The lack
of complete genome sequences for representatives of these viral subgroups has
hampered progress in the identification of viral elements correlated with host adapta-
tion associated with TMEM154 diplotypes.
Viral genomic RNA was sequenced from the lungs of clinically ill sheep at the U.S.
Meat Animal Research Center. These virus strains (USMARC-200303013-1 and USMARC-
199906011-2, from subgroups 1 and 2, respectively) were part of the original study
describing the association of SRLV genetic subgroups with TMEM154 diplotypes (8).
Encapsidated viral RNA was enriched in lung homogenates by nuclease treatment to
degrade unprotected host and environmental nucleic acids. The remaining RNA was
isolated using TRIzol LS (Life Technologies, Inc., Carlsbad, CA) and used to generate
sequencing libraries according to the RNA Iso-Seq protocol (Pacific Biosciences, Menlo
Park, CA), with one important modification. Fragment analysis revealed little RNA in the
expected range of 9 kb for complete viral genomes. Therefore, poly(A) tails were added
to the 3= ends of the RNA using a Poly(A) polymerase tailing kit (Epicentre, Madison, WI)
to allow cDNA synthesis of subgenomic fragments using the SMARTer PCR cDNA
synthesis kit (Clontech, Mountain View, CA). The resulting cDNA was amplified then size
fractionated with ELF (Sage Science, Beverly, MA). The largest fragments (approximately
2 to 3 kb) were used to generate SMRTbell libraries using the PacBio template prep kit.
The SMRTbell templates were then sequenced using Pacific Biosciences RSII sequencing
technology.
Reads were error-corrected using the SMRT Analysis server version 2.3.0 and im-
ported into the Geneious software suite (version 9.1.7; Biomatters, Auckland, New
Zealand). Adapter sequences and the added poly(A) tails were trimmed from reads
Received 2 February 2017 Accepted 3
February 2017 Published 30 March 2017
Citation Workman AM, Dickey AM, Heaton MP,
Clawson ML, Smith TPL. 2017. Complete
genome sequences of two genotype A2 small
ruminant lentiviruses isolated from infected
U.S. sheep. Genome Announc 5:e00109-17.
https://doi.org/10.1128/genomeA.00109-17.
Copyright © 2017 Workman et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Aspen M.
Workman, aspen.workman@ars.usda.gov.
VIRUSES
crossm
Volume 5 Issue 13 e00109-17 genomea.asm.org 1
  
using the BBDuk plugin (version 35.82) in Geneious. Reads greater than 1,000 nucleo-
tides in length were assembled de novo with the Geneious assembler. All reads were
then mapped to the de novo assembly, and the resulting consensus sequence was
reported. The genome sizes of USMARC-200303013-1 and USMARC-199906011-2 were
9,206 and 9,185 nucleotides, with mean read lengths of 609 and 1,099 nucleotides,
mapped read counts of 7,404 and 3,974, and mean read coverages of 617- and 721-fold,
respectively. The complete genome sequences share 87% sequence identity and
represent the first complete genome sequences for U.S. SRLV strains belonging to
genotype A2.
Accession number(s). The sequences of USMARC-200303013-1 and USMARC-
199906011-2 are deposited in GenBank/DDBJ/ENA under the accession numbers
KY358787 and KY358788, respectively.
ACKNOWLEDGMENTS
This work was supported by the Agricultural Research Service (CRIS 3040-32000-
031-00D).
We thank William Thompson, Kristen Kuhn, and Bob Lee for technical support and
Jan Watts for secretarial support.
The use of product and company names is necessary to accurately report the
methods and results; however, the U.S. Department of Agriculture (USDA) neither
guarantees nor warrants the standard of the products, and the use of names by the
USDA implies no approval of the product to the exclusion of others that may also be
suitable. The USDA is an equal opportunity provider and employer.
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Workman et al.
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Complete Genome Sequences of Two Genotype A2 Small Ruminant Lentiviruses Isolated from Infected U.S. Sheep

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Complete Genome Sequences of Two Genotype A2 Small Ruminant Lentiviruses Isolated from Infected U.S. Sheep

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