Genome organization and host range of a Brazilian isolate of johnsongrass mosaic virus

Abstract

This work reports the complete genome sequence, production of a polyclonal antiserum, and host range of a Brazilian strain of johnsongrass mosaic virus (JGMV) found infecting Panicum maximum in the state of São Paulo, Brazil. The complete genome sequence of this potyvirus, comprising 9874 nucleotides, showed 82 % amino acid sequence identity in the polyprotein to that of an isolate of JGMV from Australia. The experimental host range of this virus included mainly fodder species. Cultivated species such as rice, oats, sugarcane, rye, corn and wheat were not infected, suggesting that current isolates of this potyvirus do not represent a threat to these crops in Brazil.

Full text

BRIEF REPORT
Genome organization and host range of a Brazilian isolate
of johnsongrass mosaic virus
Viviana Marcela Camelo-Garcı
´a
1
•So
´nia Cristina da Silva Andrade
2
•
Andrew D. W. Geering
3
•Elliot Watanabe Kitajima
1
•Jorge A. M. Rezende
1
Received: 22 August 2015 / Accepted: 25 January 2016
ÓSpringer-Verlag Wien 2016
Abstract This work reports the complete genome
sequence, production of a polyclonal antiserum, and host
range of a Brazilian strain of johnsongrass mosaic virus
(JGMV) found infecting Panicum maximum in the state of
Sa
˜o Paulo, Brazil. The complete genome sequence of this
potyvirus, comprising 9874 nucleotides, showed 82 %
amino acid sequence identity in the polyprotein to that of
an isolate of JGMV from Australia. The experimental host
range of this virus included mainly fodder species. Culti-
vated species such as rice, oats, sugarcane, rye, corn and
wheat were not infected, suggesting that current isolates of
this potyvirus do not represent a threat to these crops in
Brazil.
Johnsongrass mosaic virus (JGMV; genus Potyvirus) was
first reported in Australia as maize dwarf mosaic virus
(MDMV) [24]. Until the early 1990s, the known distribu-
tion of JGMV was limited to Australia and the United
States of America, where it infects sorghum (Sorghum
bicolor), corn (Zea mays) and various weedy grasses [4,
20]. Subsequently, the presence of this virus was reported
in Colombia, Venezuela, and Nigeria [9,14,19]. In 2013,
JGMV was first detected in Brazil in Pennisetum pur-
pureum from the State of Bahia based on nucleotide
sequencing of the CP gene [22].
Leaf samples of Panicum maximum cv. Mombac¸a
exhibiting mosaic symptoms, collected in Sa
˜oLuizdo
Paraitinga, Sa
˜o Paulo state, were received by the Phy-
topathological Clinic of ESALQ/USP, Piracicaba, SP, for
diagnosis. A virus was mechanically transmitted to healthy
P. maximum cv. Mombac¸a in a greenhouse for virus
purification and host-range studies.
To determine the host range of the virus, different
species/varieties of Poaceae (Table 1) were grown from
seed and mechanically inoculated c. 30 days after germi-
nation and then again 7 days later. The original host spe-
cies was always included as an inoculation control, and
mock inoculations of each tested species were also done.
Approximately 30 days after inoculation, the plants were
inspected for the presence of symptoms, and samples from
newly emerged leaves were collected for virus testing by
plate-trapped antigen (PTA)-ELISA.
Virus purification was done using a protocol developed
for lettuce infectious chlorosis virus [6]. The concentra-
tion of purified virus was evaluated by UV spectropho-
tometry at 260 and 280 nm, using 2.8 as the extinction
coefficient. Negatively stained (1 % uranyl acetate or 1 %
sodium silicotungstate) particles were viewed under a
JEOL 1011 transmission electron microscope (JEOL,
Tokyo, Japan). For ultrastructural studies, small pieces of
infected P. maximum cv. Mombac¸a leaves were fixed,
post-fixed with OsO
4
, dehydrated, embedded in epoxy
resin, sectioned, and stained as described by Mota et al.
[15].
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-016-2772-4) contains supplementary
material, which is available to authorized users.
&Jorge A. M. Rezende
jrezende@usp.br
1
Departamento de Fitopatologia e Nematologia, Escola
Superior de Agricultura Luiz de Queiroz, Universidade de
Sa
˜o Paulo, Piracicaba, SP, Brazil
2
Departamento de Gene
´tica e Biologia Evolutiva, Instituto de
Biocie
ˆncias, USP, Sa
˜o Paulo, SP, Brazil
3
Queensland Alliance for Agriculture and Food Innovation,
The University of Queensland, GPO Box 267, Brisbane,
QLD 4001, Australia
123
Arch Virol
DOI 10.1007/s00705-016-2772-4

Table 1 Reaction of different
species/varieties of Poaceae
mechanically inoculated with a
Brazilian isolate of
johnsongrass mosaic virus
Code Species/variety Infected plants/inoculated plants
IAC5 Avena sativa 0/4
BRABR-51 Brachiaria brizantha Stapf 1/4
BRADC-13 Brachiaria decumbens Stapf 3/4
BRADC-12 Brachiaria plantaginea Hitchc. 4/4
BRARU-85 Brachiaria ruziziensis Hitchc. 0/4
CCHEC-16 Cenchrus echinatus L. 1/4
DIGHO-21 Digitaria horizontales Willd. 0/4
DIGIN-22 Digitaria insulares (L.) Fedde 0/4
ECHCO-25 Echinochloa colona (L.) Link 8/8
ECHCG-23 Echinochloa crus-galli (L.) P. Beauv. 1/4
ECHCV-24 Echinochloa crus-pavonis (Kunth) Schult. 4/4
ELEIN-26 Eleusine indica Gaertn. 0/4
ERAPI-87 Eragrostis pilosa (L.) P. Beauv. 0/4
BR2 Hordeum vulgare L. 0/8
LOLMU-83 Lolium multiflorum Lam. 0/4
MILMI-89 Melinis minutiflora P. Beauv. 4/4
ORYSA-35 Oryza sativa L. 0/4
PANMA-36 Panicum ma´ximum Jacq.cv. Colonia
˜o 0/4
ESALQ1 Panicum maximum cv. Colonia
˜o 4/8
Pennisetum purpureum 0/4
PESSE-57 Pennisetum setosum Raddi 2/3
RHYRE-41 Rhynchelytrum repens (Willd.) C.F. Hubb. 4/4
ROOEX-64 Rottboellia exaltata L.f. 3/4
CTC2 Saccharum spp. 0/4
CTC4 Saccharum spp. 0/8
CTC9 Saccharum spp. 0/4
CTC15 Saccharum spp. 0/4
CTC17 Saccharum spp. 0/4
CTC21 Saccharum spp. 0/4
CTC24 Saccharum spp. 0/4
BRSSerrano Secale cereale L. 0/8
SETGE-53 Setaria geniculata P. Beauv. 0/4
SORAR-77 Sorghum arundinaceum Roem. & Schult 0/5
CTC-S1 Sorghum bicolor (L.) Moench 0/4
201020110 Sorghum bicolor 0/4
201020122 Sorghum bicolor 0/4
201020125 Sorghum bicolor 0/4
BRS 310 Sorghum bicolor 0/4
BRS 330 Sorghum bicolor 0/4
BRS 332 Sorghum bicolor 1/4
BRS 501 Sorghum bicolor 0/4
BRS 506 Sorghum bicolor 0/4
BRS 508 Sorghum bicolor 0/4
BRS 509 Sorghum bicolor 1/4
BRS 511 Sorghum bicolor 0/4
BRS 655 Sorghum bicolor 0/4
BRS 800 Sorghum bicolor 9S. sudanense 0/8
BRS 802 Sorghum bicolor 9S. sudanense 2/8
CTC-S2 Sorghum bicolor var. sudanense 0/4
EMBRAPA1 Sorghum halepense (L.) Pers. 0/8
V. M. Camelo-Garcı
´a et al.
123

Polyclonal antibodies were obtained by intramuscular
injection of purified viral preparations emulsified with
complete (first injection) and incomplete Freund’s adjuvant
(1:1) in the thigh of a 4-month-old New Zealand female
rabbit. Four injections were done at weekly intervals, using
500-lL emulsions containing 100 lg of purified JGMV.
One week after the final injection, blood was harvested by
incisions made in the margin of the ear. The serum was
separated from clotted blood by centrifugation at 5000gfor
10 min, and then stored at -20 °C.
Plate-trapped antigen (PTA)-ELISA [16] was used to
confirm infection in host-range studies and to examine
serological relationships to sugarcane mosaic virus
(SCMV), the prevalent potyvirus infecting some cultivated
species of poaceae in Brazil. For detection, the polyclonal
antiserum was diluted 1:1000 in phosphate-buffered saline
containing 0.1 % Tween 20, 2 % PVP, MW 44,000, and
0.2 % bovine serum albumin. All samples were tested in
duplicate wells. Absorbance at 405 nm was measured
using a Metertech R960 (Metertech, Taipei, Taiwan) plate
reader, and a sample was considered positive when the
mean absorbance was greater than three times that of the
healthy control for each species.
RNA was extracted from 200 lL of purified virus using
a PureLink Viral RNA/DNA Kit (Invitrogen Carlsbad, CA,
USA). Reverse transcription was done with a High
Capacity cDNA Reverse Transcription Kit (Life Tech-
nologies), and a cDNA library was prepared using an
Illumina TruSeq SBS Kit v3-HS (200 cycles). Insert size
was estimated using an Agilent Bioanalyzer 2100 (Agilent,
Santa Clara, CA, USA) and quantified using a KAPA
Library Quantification Kit (KAPA Biosystems, Foster City,
CA, USA). The sample was barcoded and sequenced in a
HiSeq 2500 (Illumina San Diego, CA, USA) at the Center
of Functional Genomics (ESALQ/USP, Piracicaba, SP).
Read quality filtering was performed using SeqyClean
1.8.10 (https://bitbucket.org/izhbannikov/seqyclean/) using
a Phred quality score of 26 for the maximum average error,
and sequences from the Univec database (https://www.
ncbi.nlm.nih.gov/tools/vecscreen/univec/) were use as a
guide to remove possible contaminants. The filtered reads
were used for de novo assembly performed using VICUNA
v1.3 [28] with the following parameters: the kmer size was
15 bp, with minimal span of 80 bp. The contigs should
have at least 90 % similarity to be assembled together. The
maximum divergence allowed among reads to be added to
the consensus sequence was 3 %. The identity of the
consensus sequence was determined using BLASTn (Basic
Local Alignment Search Tool) from the BLAST ?suite
[3,5]. Polyprotein cleavage sites were identified manually
Table 1 continued Code Species/variety Infected plants/inoculated plants
SORHA-67 Sorghum halepense 0/12
ESALQ2 Sorghum spp. 0/8
ESALQ3 Sorghum spp. 0/8
ESALQ4 Sorghum spp. 0/8
ESALQ5 Sorghum spp. 0/8
CTC-S3 Sorghum spp. 0/4
CMSXS902 Sorghum sudanense Stapf 0/4
TX2784 Sorghum sudanense 0/4
TX2785 Sorghum sudanense 0/4
EMBRAPA2 Sorghum verticilliflorum Stapf 8/8
CD108 Triticum aestivum L. 0/8
BRS203 Triticum spp. 9Secalecereale 0/8
2B710HR Zea mays L. 0/8
3OE35H Zea mays 0/8
WXA504 Zea mays 0/8
DKB390YGRR2 Zea mays 0/8
DKB390 Zea mays 0/8
AF428 Zea mays 0/8
Tropical plus Zea mays 0/8
Pipoca Zea mays 0/8
51/384
Evaluation by symptoms and PTA-ELISA
Brazilian isolate of johnsongrass mosaic virus
123

using existing cleavage sites for members of the family
Potyviridae [1]. Deduced amino acid sequences were
obtained using ExPASy (http://ca.expasy.org/tools/dna.
html).
Sequence alignments were done using MAFFT v7.0
[11]. Phylogenetic relationships were inferred using the
maximum-likelihood method as implemented in RAxML
v.7.7.5 with one search and 100 bootstrap replicates [23].
Recombination analyses were done using the methods
included in the RDP 4.63 package with the stepdown
correction [12].
Primers to amplify the capsid protein (CP) gene were
designed based on the complete nucleotide sequence of this
Brazilian isolate of JGMV. The forward and reverse pri-
mers JGMV-F (50-CAAAGCCCCATACTTGTCGG-30)
and JGMV-R (50-TCAGACTTGGTCAGTCATCC-30)
corresponded to nt 8343-8363 and 9444-9464 in the virus
genome, respectively, and yielded a 1,121-bp amplicon.
Total plant RNA was extracted using the method of Toth
et al. [26]. RT-PCR was done in a 25-lL final volume
containing 5 lL of total RNA, 12.5 lLof29PCR Master
Mix (Thermo Fisher Scientific), 0.4 lL of each primer at
100 mM concentration, 0.04 U of AMV reverse tran-
scriptase (Promega, Madison, WI, USA) and 0.4 U of
RNase inhibitor (Ambion, Austin, TX, USA). Thermal
cycler conditions were one cycle at 42 °C for 30 min and
one cycle of 94 °C for 3 min, followed by 30 cycles at
94 °C for 30 s, 58 °C for 45 s, and 72 °for 45 s, and a final
extension at 72 °C for 10 min. The amplicons were ana-
lyzed in a 1 % agarose gel and visualized with SYBR
Ò
Safe DNA Gel Stain (Invitrogen). Direct sequencing was
done at Macrogen (Seoul, South Korea) using the ampli-
fication primers.
Leaf extracts from the original P. maximum cv. Mom-
bac¸a plants, when examined under the transmission elec-
tron microscope, had many elongated flexuous particles of
13 9700-800 nm, suggesting infection by a potyvirus.
Lamellar inclusions of type I, according to classification of
Edwardson [7], were observed in the cytoplasm, as is
characteristic of SCMV and MDMV infections. The new
antiserum was tested against homologous antigen (1:1000)
in PTA-ELISA, and it gave a positive reaction with purified
virus (A
405 nm
=1.190, 5.41 times the negative control)
and extracts from infected P. maximum cv. Mombac¸a
plants (A
405 nm
=0.781, 5.17 times the negative control)
but did not cross-react with SCMV (A
405 nm
=0.232, 1.25
times the negative control).
Sixteen of 70 species/varieties from the family Poaceae
were systemically infected with the new virus (Table 1and
Supplementary Table 1). The main symptom in all species
was a leaf mosaic, but R. repens also exhibited stunting.
The majority of members of the tribe Andropogoneae (e.g.
Saccharum spp., Zea mays,S. halapense) were resistant to
infection. Furthermore, when cultivated sorghum (S.
bicolor and hybrids) was challenged, only three of 16
genotypes became infected, and then, only small propor-
tions of the test plants. The only Sorghum spp. that was
highly susceptible to infection was S. verticilliflorum. All
of the winter cereals tested (wheat, barley and oats) were
resistant to infection.
A total of 5,770,235 single-end reads were obtained by
Illumina sequencing, of which 65 % (3,784,066 reads) were
retained after filtering. Only one contig over 3,000 bp was
recovered following the extension step. In the final contig
assembly, 1,972,159 million reads were used. Coverage var-
ied across thegenome, between 3 and 3935 per base, and with
an average of 1109and a median of 539. The complete viral
genome was 9874 nt long (Supplementary Figure 1) and has
been deposited in the GenBank database under accession no.
KT289893. One large ORF was identified spanning nt
236-9412 and, when conceptually translated, produced a
polyprotein of 3,058 amino acids. The 50and 30untranslated
regions were 235 nt and 462 nt, respectively. Domains cor-
responding to the P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-
Pro, NIb and CP were identified in the polyprotein. The
additionalopen reading frame called PIPO (‘‘pretty interesting
potyvirus ORF’’) was found at nucleotides 2797 to 3066.
Motifs considered essential for aphid transmission were found
at the N-terminus of the CP (DAG motif) and in the HC-Pro
(KTIC and PTK motifs) [18,21].
The complete nucleotide and deduced amino acid
sequences of the polyprotein were 82 % and 91.1 %
identical, respectively, to those of the type isolate of JGMV
from Australia (NC003606) (Table 2), and therefore,
according to ICTV guidelines, these isolates should be
considered conspecific. Pairwise sequence comparisons
were done for individual genes, and the P1 protein, CP and
PIPO were relatively more divergent, while the 6K1, CI
and Nib were less divergent (Table 2).
Likely sites of auto-cleavage for P1/HC-Pro and HC-
Pro/P3 are identical to those found in many potyviruses
(Supplementary Figure 1), for example, tyrosine/serine (Y/
S) and glycine/glycine (G/L), respectively [1]. The amino
acids at positions P1-P6, near the possible cleavage sites
for P3/6K1, 6K1/CI, CI/6K2, 6K2/VPg, VPg/NIa-Pro, and
NIb/CP, are identical to those described for the Australian
JGMV isolate [10]. The only difference found is for the
amino acid valine (V), at position P3 in the cleavage region
for NIa-Pro/NIb cleavage region, which is isoleucine (I) in
the Australian JGMV isolate.
A phylogenetic tree inferred using CP amino acid
sequences (Fig. 1, with final optimization likelihood
-lnL =-3920.57), clearly showed geographical segrega-
tion of clades, with virus isolates from Australia, the USA
V. M. Camelo-Garcı
´a et al.
123

and Brazil forming separate clades. Recombination analy-
sis using the entire genome sequences of MDMV
(NC003377), SCMV (NC003398), pennisetum mosaic
virus (PeMV; NC007147), sorghum mosaic virus (SrMV;
NC004035), zea mosaic virus (ZeMV; NC018833), Aus-
tralian JGMV (NC003606), and Brazilian JGMV
(KT289893) did not provide any evidence of recombina-
tion using seven independent methods available in the
RDP3 program.
Supplementary Figure 2 shows that only total RNA
extracted from some infected species of Poaceae (lines 1
through 5) were amplified by RT-PCR. The nucleotide
sequence of these fragments (924 nt) revealed 83-95 %
identity to the corresponding nucleotide sequences from
different isolates of JGMV deposited in GenBank. When
compared with the corresponding nucleotide sequences of
the isolate reported by Silva et al. [22] and the isolate of the
present work, the identities were 95 % and 100 %,
respectively.
The potyvirus isolated from P. maximum cv. Mombac¸a
was characterized by biological, serological and molecular
tests, and its identity as an isolate of JGMV was confirmed
by nucleotide sequencing of the complete genome.
According to the current species demarcation criteria for
potyviruses, virus isolates that have greater than 80 % aa
sequence identity in the CP and 76 % nt sequence identity
in the CP gene or across the entire genome are strains of
the same species [2]. Furthermore, differences in
polyprotein cleavage sites is a differentiator between
potyvirus species, and only one putative cleavage site was
found to be different between the Brazilian strain of JGMV
described in this study and that of the type strain from
Australia.
Host-range studies of JGMV isolates from different
countries have indicated a small number of susceptible
species in the monocot family Poaceae [4,20,25], as also
observed in the present work. However, there is marked
variation in the host ranges of isolates from different
countries. Among the various sorghum accessions evalu-
ated in this work, only a few were weakly susceptible to
infection (S. bicolor BRS 332, S. bicolor BRS 509 and S.
bicolor 9S. sudanense BRS 802). In comparison, Aus-
tralian isolates of JGMV readily infect sorghum unless they
carry the Krish-resistance gene, and Krish-resistance-
breaking strains have now emerged [17]. Like the Brazilian
isolate of JGMV, the isolate from Nigeria (JGMV-N) did
not infect oats and wheat although it was originally
recovered from sorghum, while a Colombian isolate from
Brachiaria spp. (JGMV-Bra) did not infect oats, sugarcane
or sorghum [14,19]. Conversely, JGMV isolates from
Venezuela and the USA, obtained from sorghum and
maize, respectively, infected oats, maize and sorghum [9,
13].
S. halepense (johnsongrass), after which the virus is
named [24], is not universally susceptible to JGMV, as
isolates from Australia, the USA and Venezuela infect this
forage, whereas isolates from Colombia and Nigeria and
from the present work did not infect this species [9,13,14,
19]. In Australia, S. halepense and S. verticilliflorum act as
perennial reservoirs of the virus [24,25], while only the
latter is susceptible to infection by the JGMV isolate
described in this study. Finally, although P. purpureum is a
host for a closely related virus isolate in the Brazilian state
of Bahia [22], this grass species was resistant to infection
using our virus isolate.
When compared to the type isolate of JGMV from
Australia, most differences in the CP of the virus isolate
described in this study occurred in the N-terminus. This
variability of the CP gene might explain some of the dif-
ferences in host range mentioned above. According to other
reports, changes in the exposed surface of the N-terminal
region of the CP have been implicated in cross-protection,
host range, and virulence [8,20]. P1 is another protein that
can play an important role in host adaptation as proposed
by Valli et al. [27]. Interestingly, the P1 protein of the
Brazilian isolate of JGMV is also very divergent when
compared to the Australian isolate of JGMV.
Table 2 Comparison of the nucleotide (nt) and deduced amino acid
(aa) sequences of johnsongrass mosaic virus isolated from P. maxi-
mum cv. Mombac¸a (KT289893) and an isolate from Australia
(NC003606)
Genome region Identity (%)
nt aa
Complete genome 82.03 -
Polyprotein - 91.06
50NTR 76.19 -
P1 72.29 74.26
HC-Pro 77.44 88.50
P3 84.44 91.07
PIPO 89.63 82.02
6K1 86.54 98.08
CI 84.10 96.78
6K2 86.16 96.23
VPg 81.66 92.06
NIa-Pro 84.37 95.44
NIb 84.20 96.52
CP 80.97 80.20
30NTR 80.20 -
Brazilian isolate of johnsongrass mosaic virus
123

At present, the JGMV found infecting P. maximum cv.
Mombac¸a does not seem to represent a threat to econom-
ically important poaceae crops such as maize, sorghum,
sugarcane, and rice in Brazil, since it was not able to infect
these species experimentally.
Acknowledgments We acknowledge Prof. Luiz L. Coutinho for the
access to computer resources for genome analysis.
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