Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

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Frequent occurrence of Mungbean yellow mosaic India virus in tomato leaf curl disease affected tomato in Oman

Scientific Reports ◽

10.1038/s41598-019-53106-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

M. S. Shahid ◽

M. Shafiq ◽

M. Ilyas ◽

A. Raza ◽

M. N. Al-Sadrani ◽

...

Keyword(s):

Rolling Circle Amplification ◽

Monopartite Begomoviruses ◽

Tomato Leaf ◽

Bipartite Begomovirus ◽

Rolling Circle ◽

Tomato Leaf Curl Disease ◽

Planting Material ◽

Next Generation Sequencing Ngs ◽

Tomato Leaf Curl ◽

Leaf Curl

Abstract Next generation sequencing (NGS) of DNAs amplified by rolling circle amplification from 6 tomato (Solanum lycopersicum) plants with leaf curl symptoms identified a number of monopartite begomoviruses, including Tomato yellow leaf curl virus (TYLCV), and a betasatellite (Tomato leaf curl betasatellite [ToLCB]). Both TYLCV and ToLCB have previously been identified infecting tomato in Oman. Surprisingly the NGS results also suggested the presence of the bipartite, legume-adapted begomovirus Mungbean yellow mosaic Indian virus (MYMIV). The presence of MYMIV was confirmed by cloning and Sanger sequencing from four of the six plants. A wider analysis by PCR showed MYMIV infection of tomato in Oman to be widespread. Inoculation of plants with full-length clones showed the host range of MYMIV not to extend to Nicotiana benthamiana or tomato. Inoculation to N. benthamiana showed TYLCV to be capable of maintaining MYMIV in both the presence and absence of the betasatellite. In tomato MYMIV was only maintained by TYLCV in the presence of the betasatellite and then only at low titre and efficiency. This is the first identification of TYLCV with ToLCB and the legume adapted bipartite begomovirus MYMIV co-infecting tomato. This finding has far reaching implications. TYLCV has spread around the World from its origins in the Mediterranean/Middle East, in some instances, in live tomato planting material. The results here may suggest that begomoviruses which do not commonly infect tomato, such as MYMIV, could be spread as a passenger of TYLCV in tomato.

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Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome

Cytogenetic and Genome Research ◽

10.1159/000509845 ◽

2020 ◽

Vol 160 (6) ◽

pp. 329-334

Author(s):

Alfredo Diaz-Lara ◽

Nola J. Mosier ◽

Kristian Stevens ◽

Karen E. Keller ◽

Robert R. Martin

Keyword(s):

High Throughput Sequencing ◽

Rolling Circle Amplification ◽

Standard Test ◽

Molecular Techniques ◽

Test Method ◽

Plant Genome ◽

Red Raspberry ◽

Rolling Circle ◽

Certification Programs ◽

Dnase Treatment

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis “Munger” has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on “Munger”, suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

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Use of RCA-PCR assay for detection of Begomovirus infection in Bhut Jolokia (Capsicum assamicum) in Tezpur region of Assam, India

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.6(2).p64-69 ◽

2016 ◽

Vol 6 (2) ◽

pp. 64-69

Author(s):

Ajitabh Bora ◽

Hemant Kumar Gogoi ◽

Mohan C. Kalita

Keyword(s):

Rolling Circle Amplification ◽

Viral Disease ◽

Viral Pathogen ◽

Rolling Circle ◽

North East India ◽

North East ◽

Detection Assay ◽

A Genome ◽

Bhut Jolokia ◽

Leaf Curl

Bhut Jolokia (Capsicum assamicum), one of the hottest chilli in the world is a chilli cultivar, endemic to North East India and is extensively cultivated in the states of Assam, Nagaland and Manipur. The demand of this chilli is very high in domestic as well as in the international market due to its extreme hotness and pleasant aroma. This plant is severely affected by leaf curl disease caused by Begomovirus, leading to total crop loss. Accurate diagnosis of this viral disease at seedling stage, prior to transplanting, is essential to prevent further spread of the disease. With an aim to device a rapid and accurate detection assay, Rolling Circle Amplification (RCA)-Polymerase Chain Reac-tion (PCR) technique was used for detection of the viral pathogen. RCA tech-nique was employed to increase the viral template and PCR was conducted using a degenerate primer pair. With the help of this assay, 1.4 kbp segment of DNA-A genome of the Begomovirus was amplified. Phylogeny revealed close proximity of the isolate with Tomato leaf curl Bangladesh virus. This is the first report on characterization of Begomovirus infecting Bhut Jolokia in Tezpur region of Assam.

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First Report of Ageratum enation virus, Betasatellite and Alphasatellite Causing Leaf Curl and Enation Disease of Amaranthus hypochondriacus in India

Plant Disease ◽

10.1094/pdis-04-14-0338-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1285-1285 ◽

Cited By ~ 6

Author(s):

A. Srivastava ◽

S. Kumar ◽

S. K. Raj

Keyword(s):

Sequence Data ◽

Disease Incidence ◽

Rolling Circle Amplification ◽

Nucleotide Identity ◽

Post Inoculation ◽

Specific Primers ◽

Rolling Circle ◽

Amaranthus Hypochondriacus ◽

Close Phylogenetic Relationship ◽

Leaf Curl

During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. E. Bull et al. Mol. Biotechnol. 23:83, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

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Identification of Chili leaf curl virus Causing Leaf Curl Disease of Petunia in Oman

Plant Disease ◽

10.1094/pdis-06-13-0678-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 572-572 ◽

Cited By ~ 5

Author(s):

A. A. Al-Shihi ◽

S. Akhtar ◽

A. J. Khan

Keyword(s):

Indian Subcontinent ◽

Rolling Circle Amplification ◽

Rapid Evolution ◽

Post Inoculation ◽

Multiple Sequence ◽

Rolling Circle ◽

Begomovirus Genome ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Petunias (Petunia × hybrida) are the most important ornamental plants in Oman. In 2012, petunias were observed in public parks and airport landscape in Dhofar region with symptoms of upward leaf curling, yellowing and vein clearing, and size reduction in leaves. Almost all plants in the surveyed landscape showed high infestation of Bemisia tabaci and symptoms that suggested infection with a begomovirus. Six symptomatic samples were collected from three different sites. All symptomatic samples were found PCR-positive with diagnostic primers for begomovirus (3) when DNA extracted from infected leaves was used as template. Nucleic acids extracted from the symptomatic leaves were used to amplify circular DNA molecules by rolling circle amplification method. The amplified concatameric products were digested with restriction enzyme PstI, which yielded a product ∼2.8 kb in size. The putative begomovirus fragment was cloned and sequenced in both orientations. Partial sequences of six clones were 99 to 100% similar and thus only two clones, PT-2 and PT-3, were fully sequenced. The whole genomes of both clones were 2,761 bp, and both were deposited in GenBank under accession numbers HF968755 and HF968756 for the isolates PT-2 and PT-3, respectively. Both sequences had six open reading frames; Rep, TrAP, REn, and C4 genes in complementary sense; and CP and V2 genes in virion-sense, typical of the begomovirus genome organization. Upon alignment, the two sequences showed 99.4% nucleotide identity with each other, thus representing isolates of a single begomovirus species. BlastN comparison showed PT-2 and PT-3 from petunia were 94 to 95% identical to the sequences of ChCLV from Oman (JN604490 to JN604500), which were obtained from other hosts. ClustalV multiple sequence alignment showed that isolates PT-2 and PT-3 shared maximum sequence identity of 93.3 and 92.8%, respectively, with an isolate of ChLCV-OM (JN604495). According to ICTV rules for begomoviruses, PT-3 should be considered to be a new strain of ChLCV-OM and PT-2 a variant of the already existing ChLCV-OM strain. We propose the name for this new strain as the “Petunia strain” of Chili leaf curl virus (ChLCV-Pet). Two infectious clones were constructed from the PT-2 and PT-3 sequences, clones as 1.75-genome sequences in a binary vector, suitable for agroinfection to confirm their infectivity. Both clones, PT-2 and PT-3, produced typical leaf curl disease symptoms upon inoculation on petunia 18 days post inoculation. The presence of the same virus in symptomatic field infected and inoculated petunia was confirmed by Southern blot using 650 bp DIG labeled probe prepared from CP region of PT-3 isolate. ChLCV-OM, a monopartite begomovirus, is widely associated with leaf curl disease of tomato and pepper in Oman, with its origin traced to the Indian subcontinent (2). Identification of a new strain of ChLCV from petunia provides evidence of an ongoing rapid evolution of begomoviruses in this region. Although petunia has been tested as an experimental host for some begomoviruses (1,4), this is the first report of petunia as natural host for ChLCV, a begomovirus previously reported in tomato and pepper in Oman. References: (1) Cui et al. J. Virol. 78:13966, 2004. (2) Khan et al. Virus Res. 177:87, 2013. (3) Khan et al. Plant Dis. 97:1396, 2013. (4) Urbino et al. Arch. Virol. 149:417, 2003.

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A workflow for accurate metabarcoding using nanopore MinION sequencing

10.1101/2020.05.21.108852 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bilgenur Baloğlu ◽

Zhewei Chen ◽

Vasco Elbrecht ◽

Thomas Braukmann ◽

Shanna MacDonald ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sequence Data ◽

Rolling Circle Amplification ◽

Error Rates ◽

Read Length ◽

Taxonomic Assignment ◽

Major Drawback ◽

Rolling Circle ◽

Sequencing Platform ◽

Sequencing Platforms

AbstractMetabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads.We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

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Cloning, Structural Characterization, and Phylogenetic Analysis of Flower MADS-Box Genes from Crocus (Crocus sativusL.)

The Scientific World JOURNAL ◽

10.1100/tsw.2007.175 ◽

2007 ◽

Vol 7 ◽

pp. 1047-1062 ◽

Cited By ~ 8

Author(s):

Athanasios S. Tsaftaris ◽

Alexios N. Polidoros ◽

Konstantinos Pasentsis ◽

Apostolos Kalivas

Keyword(s):

Molecular Mechanisms ◽

Phylogenetic Analyses ◽

Rolling Circle Amplification ◽

Crocus Sativus ◽

Flower Formation ◽

Mads Box ◽

Crop Species ◽

Rolling Circle ◽

Cdna Sequences ◽

Single Compound

Crocus (Crocus sativusL.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation.Here we review the different methods followed or developed for obtaining these sequences involving conventional 5' 3' RACE, as well as newly developed methods from our group, named Rolling Circle Amplification – RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.

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Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus

Pathogens ◽

10.3390/pathogens9030214 ◽

2020 ◽

Vol 9 (3) ◽

pp. 214

Author(s):

Samira Samarfard ◽

Alistair R. McTaggart ◽

Murray Sharman ◽

Nicolás E. Bejerman ◽

Ralf G. Dietzgen

Keyword(s):

Complete Genome ◽

High Throughput Sequencing ◽

Rna Viruses ◽

Sequence Data ◽

Rna Virus ◽

Rolling Circle Amplification ◽

French Bean ◽

Cdna Libraries ◽

Restriction Enzyme Digestion ◽

Rolling Circle

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.

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Molecular Characteristics of Jujube Yellow Mottle-Associated Virus Infecting Jujube (Ziziphus jujuba Mill.) Grown at Aksu in Xinjiang of China

Viruses ◽

10.3390/v13010025 ◽

2020 ◽

Vol 13 (1) ◽

pp. 25

Author(s):

Jiashu Guo ◽

Yanxiang Wang ◽

Guoping Wang ◽

Jian Hong ◽

Zuokun Yang ◽

...

Keyword(s):

Genetic Diversity ◽

High Throughput Sequencing ◽

Close Association ◽

Field Investigation ◽

Liaoning Province ◽

Molecular Characteristics ◽

Rt Pcr ◽

Natural Hosts ◽

Yellow Mottle ◽

Ziziphus Jujuba

Chinese jujube (Ziziphus jujuba Mill.) is a native fruit crop in China. Leaf mottle and dapple fruit disease is prevalent in cultivated jujube plants grown at Aksu in Xinjiang Uygur Autonomous Region of China. Jujube yellow mottle-associated virus (JYMaV), a tentative member in the genus Emaravirus, was recently identified from mottle-diseased jujube plants grown in Liaoning Province in China, but its incidence and genetic diversity in China is unknown. In this study, the genome sequences of three JYMaV isolates from two jujube cultivars and one jujube variant were determined by high-throughput sequencing (HTS) for small RNA and rRNA-depleted RNA coupled with RT-PCR assays. Comparison of these sequences together with sequences of the viral RNA segments derived by primer set 3C/5H-based RT-PCR revealed that genetic diversity was present in the virus populations and high sequence variation occurred at the non-translational regions of each of the viral genomic segments. Field investigation confirmed the close association of the virus with leaf mottle symptoms of jujube plants. Furthermore, this study revealed that P5 encoded in the viral RNA5 displayed a nuclear localization feature differing from the plasmodesma (PD) subcellular localization of the virus movement protein (P4), and the two proteins could interact with each other in the BiFC assays. Our study provides a snapshot of JYMaV genetic diversity in its natural hosts.

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The genetic diversity of “papillomavirome” in bovine teat papilloma lesions

Animal Microbiome ◽

10.1186/s42523-021-00114-3 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Jéssica Tatiane Sauthier ◽

Cíntia Daudt ◽

Flavio Roberto Chaves da Silva ◽

Christian Diniz Beduschi Travassos Alves ◽

Fabiana Quoos Mayer ◽

...

Keyword(s):

Genetic Diversity ◽

High Throughput Sequencing ◽

Molecular Detection ◽

Bos Taurus ◽

Rolling Circle Amplification ◽

Dna Viruses ◽

Rolling Circle ◽

Double Stranded Dna ◽

Wide Range ◽

Study Support

Abstract Background Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. Results Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse “papillomavirome” in bovine teat warts. Conclusions The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity.

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