Eur J Plant Pathol (2021) 159:203–210

https://doi.org/10.1007/s10658-020-02124-4

The complete sequence of a papaya ringspot virus (PRSV)

isolate from West Bengal, India infecting papaya and study
of genetic variation
Siriya Sultana & Buddhadeb Roy & Ang Rinzing Sherpa

Received: 30 March 2020 / Revised: 8 September 2020 / Accepted: 29 September 2020 / Published online: 6 October 2020
# Koninklijke Nederlandse Planteziektenkundige Vereniging 2020

Abstract Papaya ringspot virus (PRSV) is one of the Keywords Papaya ringspot virus . Complete genome .
important pathogens responsible for damage of the pa- Phylogeny
paya crop and causes significant yield loss of fruits and
vegetable worldwide and hence a serious threat. In this
study, PRSV-infected plant samples collected from dif-
Papaya ringspot virus (PRSV) is a definitive member of
ferent locations in North 24 Parganas districts of West
the genus Potyvirus in the family Potyviridae (Van
Bengal, were tested with potyvirus specific ELISA and
Regenmortel et al. 2000). It is one of the most devastat-
further confirmed with potyvirus group specific degen-
ing virus constraints to the papaya industry (Carica
erate primers by RT PCR, TEM and sequence analysis.
papaya L.). PRSV is transmitted to papaya and cucur-
Our result indicates that the disease symptoms occurred
bits through aphids in a non-persistent manner (Purcifull
in 80–100% of the plant in the field surveyed. The
et al. 1984), through seed and mechanical transmission
complete sequence of PRSV isolate WB was deter-
(Gonsalves et al. 2010). The virions of PRSV are flex-
mined from RT-PCR products with specific primer pairs
uous, filamentous rod measuring 760–800 nm × 12 nm
designed covering the whole genome and confirmed by
(Yeh and Gonsalves 1984) consisting ssRNA (+) sense
next-generation sequencing. The whole genome of the
genome of approximately 10,326–10,343 nucleotides.
PRSV was determined and submitted to GenBank as
PRSV has a single large open reading frame that trans-
accession no. LC482263. The sequence of PRSV-WB
lates into a large polypeptide and subsequently cleaved
(10,340 bp) was found to share upto 90% and 93%,
into 10 functionally active proteins (Yeh et al. 1992;
identity with other PRSV isolates at the nucleotide and
Wang and Yeh 1997). The isolates of PRSV are broadly
amino acid levels respectively. Based on phylogenetic
classified into two biotypes, those that infect papaya are
analysis, PRSV- WB was found to be most closely
PRSV-P type and the cucurbit infecting isolates are
related to PRSV-BD2 (MH397222) from papaya in
termed as PRSV-W (Yeh et al. 1984). In India, PRSV
Bangladesh (geographically close).
in papaya was first reported in 1958 from the western
part of India, later it spread over many geographical
regions including Himachal Pradesh, Chhattisgarh,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10658-020-02124-4) contains Jharkhand, Delhi, Karnataka, Uttar Pradesh, Maharash-
supplementary material, which is available to authorized users. tra (Jain et al. 2004) and Tamil Nadu (Sharma et al.
2005), causing several crop losses of up to 85–90%
S. Sultana : B. Roy : A. R. Sherpa (*) (Lokhande et al. 1992; Hussain and Varma 1994). To
Department of Botany, West Bengal State University, Malikapur,
Berunanpukuria, 24 North Parganas, Barasat, Kolkata, W.B.
develop any virus control strategies, first it is essential to
700126, India understand the genetic variation and structure of the
e-mail: ang2001@gmail.com virus population. Most of the genetic variation studies
204
were done based on the sequence of coat protein and 3′
region of PRSV isolates (Silva-Rosales et al. 2000; Jain
et al. 2004; Wei et al. 2007; Kumar et al. 2019) and not
much attention was given to the whole genome se-
quences. This report focus on the disease incidence
and the determination of the complete genome of PRSV
isolate from West Bengal. In order to determine the
sequence variability of PRSV isolates from different
geographical locations of world, all complete genome
of PRSV isolates including our sequence were analyzed
both at nucleotide and amino acid sequences level.
During a survey in the year 2016–2018, North 24
Parganas, West Bengal, leaf samples were collected
from papaya plants showing typical symptoms of PRSV
(leaf mosaic, vein clearing, leaf distortion resulting into
shoe-string, yellow mottling, stunted growth of plant
and fruit malformation). The collected samples were
immediately stored in RNA later® solution
(Thermofisher) and brought to the plant virology labo-
ratory for downstream identification and characteriza-
tion. The identity of the virus was confirmed by patho-
genicity tests on Carica papaya. For further confirma-
tion of PRSV in papaya, potyvirus group specific
enzyme-linked immunosorbent assay (ELISA) (Agdia,
USA), TEM, mechanical transmission study and RT-
PCR using universal degenerate primer pair: MJ1 and
MJ2 (Marie-Jeanne et al. 2000) were used.
In this study occurrence of Papaya ringspot virus like
disease symptoms is found in 80–100% of the plants
surveyed. Besides C. papaya and Cucurbits, many
plants of viz Clitoria ternatea, Coccinia grandis were
found to be infected with the PRSV. This finding indi-
cates that these plants may acts as reservoir for the
PRSV. A detail survey and identification of reservoir
hosts are essential for the study and management of
virus diseases. All the 48 collected samples with
PRSV-like symptoms were positive for PRSV by
ACP-ELISA and RT-PCR. Total RNA was extracted
from ELISA positive fresh leaf sample by Trizol (Sig-
ma, USA) according to manufacturer’s instruction and
used directly as a template for cDNA synthesis. In the
present investigation different sets of PRSV specific
primer pairs (Supplementary Table 1) were designed
to amplify the whole genome of PRSV isolate WB.
Sequence was further confirmed by next-generation
sequencing (NGS) analysis of the infected plant on
Illumina platform.
To check the identity and variability of the PRSV
isolates from India and other geographical regions of the
Eur J Plant Pathol (2021) 159:203–210
world; BLAST, multiple sequence alignment, and phy-
logenetic trees were constructed both at nucleotide as
well as at amino acid sequence level. The complete
genome sequences of PRSV isolates were retrieved
from the GenBank from different regions (Table 1).
For genetic variability analyses, multiple sequences
alignment of all complete genomes was done with the
ClustalW algorithm (Larkin et al. 2007) implemented in
the program MEGA 7.0 (Kumar et al. 2016).
The complete genome sequence of PRSV isolate WB
was determined by joining overlapping sequences ob-
tained from RT-PCR products with different sets of
primer pairs and further confirmed by next-generation
sequencing (or deep sequencing) and was deposited in
GenBank as Accession no. LC482263. The genome is
10, 340 nucleotides long encoding a polyprotein of 3342
amino acid residues. BLASTn showed nucleotide iden-
tities between 81.34 to 90.82% between the PRSV
isolate WB and worldwide PRSV isolates whose ge-
nomes were completely sequenced. The highest nucle-
otide identity (90.82%) was with PRSV isolate BD-2
from Bangladesh (MH397222) which is geographically
close to India (Hamim et al. 2019) and the lowest
nucleotide identity (81.34%) was with PRSV-China
(KY933061). At amino acid (aa) level (BLASTp),
PRSV-WB exhibited the lowest amino acid identity
(87%) with PRSV-SK (KY996464) and the highest
amino acid identity (93.66%) with BD-2 isolate from
Bangladesh (MH397222). The P1 protein was the most
variable (57.92–83.26%), while 3’-UTR regions of
PRSV are highly conserved.
Pairwise sequence identity analysis was carried out
using the Sequence Demarcation Tools (SDT) version
1.2 with default parameters to obtain the colour coded
matrix of the scores. The nucleotide identity of complete
genome sequence of PRSV-WB was analyzed and com-
pared with reported complete genome sequences of the
PRSV isolates from different parts of the world (Fig. 1).
Nucleotide identity of PRSV with other PRSV isolates
ranged from 75 to 90%, being the highest nucleotide
identity (90.72) was with PRSV BD-2 isolate from
Bangladesh (MH397222) and lowest nucleotide identity
(75.18%) was with PRSV BD-1 isolate from Bangla-
desh (MH444652).
The phylogenetic tree (Fig. 2) was based on nucleo-
tide sequences of the PRSV- WB isolate and other
PRSV isolates reported worldwide and available in
GenBank. The PRSV isolates were found to be clustered
into three major clades or groups, Clade I, Clade II and
Eur J Plant Pathol (2021) 159:203–210 205

Table 1 Origins, hosts, and accession numbers of Papaya ringspot virus isolates/strains whose complete genome sequences were used in
this study

Origin Isolates/strains Host Accession number

China(9) XM, Hn, PRSV-SD Carica papaya, Cucurbita pepo, KT895257, KT633943, KF734962,
HN-DF HAINAN Pumpkin HQ424465, KF791028,
MF085000, MF074214,
KY933061, EF183499
India (12) P, HYD, PM-H, PM-I, PRSVR3, Vasconcellea cauliflora, LC482263, MF356497,
Pune (Aundh) VC,W, Carica papaya MH311882, EF017707,
PS3-H, PS3-I KP743981, MF405295,
MF405296, KJ755852,
EU475877, MF405299,
MF405297, MF405298
East Timor (4) SK, W Cucurbits KX655874, KX655865,
KX655864, KX655863
Australia (13) W Curcurbits KX998708, KX998707,
KX655866, KX655867,
KX655868, KX655869,
KX655871, KX655861,
KX655860, KX655862,
KX655872, KX655873
USA (3) W, PG, PRSV-ptx Curcurbit, Gourd, Carica papaya KY271954, EU126128, KY039583
Brazil (3) W Cucurbita pepo, Fevillea cordifolia DQ374153, DQ374152,
Taiwan (13) W, P, PRSV-SMN, Luffa cylindrica, Carica papaya NC_001785, X97251, AY027810,
PRSV-DF, HA JX448372, JX448369,
JX448370, JX448373,
JX448371, DQ340769,
DQ340770, DQ340771, X67673,
EU882728
Thailand (2) W Not mention AY010722, AY162218
France (1) E2 Cucurbita pepo KC345609
Mexico (1) Mex-VrPO Not mention AY231130
South Korea (2) Watermelon strain Cucumber, Cucurbita pepo KY996464, AB369277
Colombia (2) P Carica papaya Maradol KT275938, KT275937
Bangladesh (2) BD-1, BD-2 Papaya MH397222, MH444652

Clade III. PRSV-WB was in Clade II, with all Indian
PRSV isolates and Bangladesh PRSV isolates
(MH397222). All Australian and American PRSV iso-
lates formed a distinct cluster (Clade III). PRSV-WB
formed a minor group with PRSV-Bangladesh
(MH397222) and PRSV- Meghalaya (MF356497) and
PRSV- Pune (MH311882).
Recombination Detection Program RDP4 version
Beta 80 was used to detect and analyze the recombina-
tion events between the PRSV isolates (Martin et al.
2015) using seven methods, include RDP,
GENECONV, BootScan, MaxChi, Chimaera, SiScan,
and 3Seq with their default parameters to find out the
recombinant isolates and a P value threshold of 0.05.
Only those events recognized by at least three different
algorithms were admitted as evidence for recombina-
tion. Twenty-four recombination break point events
were detected in 22 PRSV genomes at multiple regions
when all the complete genome of the PRSV isolates
were analyzed. Recombination events in Indian, Chi-
nese, and American isolates were identified, when the
p values obtained were less than 1 × 10−6 for at least
three out of seven approaches (Fig. 3, Supplementary
Table 2). According to previous studies, it appears that
recombination occurs repeatedly in the Potyvirus genus.
Thus, recombination is one of the most important factors
that allowed potyviruses to adapt to various hosts and
different environments in order to survive and spread
206
Fig. 1 Pairwise nucleotide sequence identity matrix colour plot of the 66 PRSV isolates/strains from the GenBank based on the complete
genome using SDT v1.2software
(Zhou et al. 2014). Our data showed that PRSV isolates
from Asian countries especially India and China show
more recombinants than other countries. One of the
major mechanisms in the evolution of PRSV is recom-
bination, which may have a role in maintaining the
effectiveness of purifying selection by preventing the
accumulation of deleterious mutations. Recombination
has been associated with the expansion of host range,
the modification of transmission vector specificity, the
evolution of new viruses, increase in virulence and
pathogenesis and the evasion of host immunity (Noa-
Carrazana et al. 2007).
According to NCBI database, more than 66 complete
genome sequences and approximately 2635 partial ge-
nome sequence of PRSV isolates are available from
different parts of the globe. From India, until now, 11
complete genome sequences and 313 partial gene
Eur J Plant Pathol (2021) 159:203–210
sequences of PRSV are available (up to November,
2019). The nature of PRSV population is highly diverse
making significant constraint to papaya disease manage-
ment (Jain et al. 2004). Coat Protein-Mediated Resis-
tance (CPMR) has been successfully used to confer
resistance to a wide range of viruses including PRSV
(Lomonossoff 1995; Gonsalves 1998). The CP mediat-
ed PRSV-resistant transgenic papaya cultivars, the Sun-
Up and Rainbow has been commercially grown in Ha-
waii since 1998, authorized for marketing and have
played the most major role in saving the papaya industry
from economic demise.
In the current study, genetic variability and phyloge-
netic relationship of all PRSV isolates based on the
complete genomes was determined. Phylogenetic anal-
ysis of the whole genome sequences of the PRSV-WB
isolates from this study with other PRSV isolates from
Eur J Plant Pathol (2021) 159:203–210 207

Fig. 2 Phylogenetic tree

generated from maximum
likelihood analysis using a
GTR + G model of nucleotide
substitution (−lnL =
−(−120,323.15)) based on
complete genome of 66 PRSV at
nucleotide level using MEGA 7
software. Onion yellow dwarf
virus (OYDV) (Accession no.
AB219834) sequence was used as
Out group, marked with black
square and our sequence
(Accession no. LC482263)
marked with red triangle. No.
below the branches indicates
bootstrap value percentages from
1000 replications, but bootstrap
support values above 50% are
shown

different parts of the world shows three distinct clusters.
According to the International Committee on Taxono-
my of Viruses (ICTV), the species demarcation criteria
for complete open reading frame (ORF) different spe-
cies within the genus Potyvirus requires nucleotide se-
quence identity to be <76% and the amino acid se-
quence identity <82% for species to be distinct
(Adams et al. 2005).
It is suggested that while designing transgenes for
potyvirus resistance, it is essential to select regions of at
least 90% identity between isolates to obtain a wide
resistance (Moreno et al. 1998). In India, the occurrence
of PRSV has been relatively new in comparison to other
countries and coincides with the sudden increase in
papaya cultivation and the introduction of new varieties
from different geographical regions, which may exert
different levels of selection pressure on the virus. Since,
selection of transgenes is crucial for development of
virus resistance transgenic papaya in India, availability
of more complete genome sequences of PRSV from the
Indian sub-continent will help a better assessment of the
sequence divergence within the PRSV population. The
208 Eur J Plant Pathol (2021) 159:203–210

Fig. 3 Regions of recombination events in the PRSV populations based on the complete genome by using RDP 4 version Beta 80 software
Eur J Plant Pathol (2021) 159:203–210
present study supports the previous reports that PRSV
shows frequent recombination and high genetic varia-
tion. Bateson et al. 2002 discussed that mutation, to-
gether with local and long-distance movement, contrib-
utes to population variation, and also discussed that
populations of the PRSV-P biotype have evolved on
several occasions from PRSV-W populations. They also
suggested that even where divergence in PRSV-P is
low, highly divergent cucurbit populations may act as
potential reservoirs of new P sequences. For effective
disease management and the development of PRSV
resistant transgenic papaya, it is essential to survey,
detect and characterize the PRSV population in different
papaya growing regions.
Acknowledgements Authors would like to thank Department of
Science and Technology, New Delhi for SERB Project Grant No.
SR/FT/LS-165/2012 and financial assistant from University
Grants Commission (UGC), New Delhi to Miss Siriya Sultana,
Senior Research Fellow (Maulana Azad National Fellowship) in
the form of fellowship is greatly acknowledged. Authors also
acknowledge the WB DST supported FIST and Boost program
for the facilities to carry out the experiments.
Funding The work of S.S. was supported by University Grants
Commission (UGC), New Delhi as Senior Research Fellowship
under Maulana Azad National Fellowship, and the work from
A.R.S was funded by Department of Science and Technology,
New Delhi for SERB Project Grant No. SR/FT/LS-165/2012.
Compliance with ethical standards
Conflict of interest The authors declare that they have no con-
flict of interest.
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors
References
Adams, M. J., Antoniw, J. F., & Fauquet, C. M. (2005). Molecular
criteria for genus and species discrimination within the family
Potyviridae. Archives of Virology, 150, 459–479. https://doi.
org/10.1007/s00705-004-0440-6.
Bateson, M. F., Lines, R. E., Revill, P., Chaleeprom, W., Ha, C.
V., Gibbs, A. J., & Dale, J. L. (2002). On the evolution and
molecular epidemiology of the potyvirus papaya ringspot
virus. Journal of General Virology, 83, 2575–2585.
Gonsalves, D. (1998). Control of Papaya ringspot virus in papaya:
A case study. Annual Review of Phytopathology, 36, 415–
437.
Hamim, I., Rwahnih, M. A., Borth, W. B., Suzuki, J. Y., Melzer,
M. J., Wall, M. M., Green, J. C., & Hu, J. S. (2019). Papaya
209
ringspot virus isolates from papaya in Bangladesh: Detection,
characterization, and distribution. Plant Disease, 103, 2920–
2924. https://doi.org/10.1094/PDIS-12-18-2186-RE.
Hussain, S., & Varma, A. (1994). Occurrence of Papaya ringspot
virus (PRSV) in Amritsar (Punjab) India. Journal of
Phytopathological Research, 7, 77–78.
Jain, R. K., Sharma, J., Sivakumar, A. S., Sharma, P. K., Byadgi,
A. S., Verma, A. K., & Varma, A. (2004). Variability in the
coat protein gene of Papaya ringspot virus isolates from
multiple locations in India. Archives of Virology, 149,
2435–2442. https://doi.org/10.1007/s00705-004-0392-x.
Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular
evolutionary genetics analysis version 7.0 for bigger datasets.
Molecular Biology and Evolution, 33, 1870–1874.
Kumar, R., Srivastava, A., Srivastava, A., Srivastava, S., Prasad,
V., & Raj, S. K. (2019). Studies on molecular variability of
coat protein gene of Papaya ringspot virus-P isolates from
India. Europian Journal of Plant Pathology, 155, 369.
https://doi.org/10.1007/s10658-019-01778-z.
Larkin, M. A., Blackshields, G., Brown, N. P., & Chenna, R.
( 2 0 0 7 ) . C l u s t a l W a n d C l u s t a l X v e r si o n 2 . 0 .
Bioinformatics, 23, 2947–2948.
Lokhande, N. M., Moghe, P. G., Matte, A. D., & Hiware, B. J.
(1992). Occurrence of papaya ringspot virus (PRSV) in
Virdharbha region of Maharastra. Journal of Soils crops, 2,
36–39.
Lomonossoff, G. P. (1995). Pathogen derived resistance to plant
viruses. Annual Review of Phytopathology, 33, 323–343.
Marie-Jeanne, V., Ioos, R., Peyre, J., Alliot, B., & Signoret, P.
(2000). Differentiation of Poaceae potyviruses by reverse
transcription–polymerase chain reaction and restriction anal-
ysis. Journal of Phytopathology, 148, 141–151.
Martin, D. P., Murrell, B., Golden, M., Khoosal, A., & Muhire, B.
(2015). RDP4: Detection and analysis of recombination pat-
terns in virus genomes. Virus Evolution, 1(1), vev003.
https://doi.org/10.1093/ve/vev003.
Moreno, M., Bernal, J. J., Jimenez, I., & Rodriguez-Cerezo, E.
(1998). Resistance in plants transformed with the P1 or P3
gene of tobacco vein mottling potyvirus. Journal of General
Virology, 79, 2819–2827.
Noa-Carrazana, J. C., Gozalez-de-Leon, D., & Silva-Rosales, L.
(2007). Molecular characterization of a severe isolates of
Papaya ringspot virus in Mexico and its relationship with
other isolates. Virus Genes, 35, 109–117.
Purcifull, D. E., Edwardson, J. R., Hiebert, E., & Gonsalves, D.
(1984). Papaya ringspot virus. Descriptions of plant viruses.
AAB. No. 292.
Sharma, J., Jain, R. K., Ramiah, M., & Varma, A. (2005). Natural
spread of Papaya ringspot virus to new areas: Occurrence in
Coimbatore, Tamil Nadu. Indian Phytopathology, 58, 245–
249.
Silva-Rosales, L., Ruiz-Castro, S., Teliz-Ortiz, D., & Noa-
Carrazena, J. C. (2000). Coat protein sequence comparison
of three Mexican isolates of Papaya ringspot virus with other
geographical isolates reveal a close relationship to American
and Australian isolates. Archives of Virology, 145, 835–843.
Van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L.,
Carstens, E.B., Estes, M.K., Leman, S.M., Maniloff, J.,
Mayo, M. A., McGeoch, D. J., Pringle, D. J., & Wickner,
R.B. (2000). Virus taxonomy. Classification and nomencla-
ture of viruses. Seventh Report of the International
210
Committee on Taxonomy of Viruses. Academic Press, San
Diego.
Wang, C. H., & Yeh, S. D. (1997). Divergence and conservation
of the genomic RNAs of Taiwan and Hawaii strains of
papaya ringspot potyvirus. Archives of Virology, 142, 271–
285.
Wei, J., Liu, D., Li, X., & Zhou, P. (2007). Variation in the coat
protein gene of Papaya ringspot virus isolates from multiple
locations of China. Journal of IntegrativePlant Biology, 49,
1062–1069.
Yeh, S. D., & Gonsalves, D. (1984). Purification and immunolog-
ical analysis of cylindrical inclusion protein induced by
Papaya ringspot virus and Watermelon mosaic virus-1.
Phytopathology, 74(11), 1273–1278.
Eur J Plant Pathol (2021) 159:203–210
Yeh, S. D., Gonsalves, D., & Provviddenti, R. (1984).
Comparative studies on host range and serology of Papaya
ringspot virus and Watermelon mosaic virus.
Phytopathology, 74, 1081–1085.
Yeh, S. D., Jan, F. J., Chiang, C. H., Doong, T. J., Chen, M. C.,
Chung, P. H., & Bau, H. J. (1992). Complete nucleotide
sequence and genetic organisation of papaya virus RNA.
Journal of General Virology, 73, 2531–2541.
Zhou, G. C., Wu, X., Zhang, Y. M., & Wu, P. (2014). A genomic
survey of thirty soybean–infecting Bean common mosaic
virus (BCMV) isolates from China pointed BCMV as a
potential threat to soybean production. Virus Research, 191,
125–133.