First Report of the Occurrence of Banana streak Goldfinger Virus (BSGFV)in the State of Minas Gerais, Brazil
Farias ARG, Figueira AR*, Geraldino PS and Pompeu DC
Departamentof Phytopathology, Federal Universityof Lavras -MG, Brazil
Submission: October 02, 2018; Published: September 04, 2018
*Corresponding author: Figueira AR, Departament of Phytopathology, Molecular Virology Laboratry, Federal Universityof Lavras -MG–Brazil, Tel: +55-(35)-3829-1282; E-mail: antoniarfigueira@gmail.com
How to cite this article: Farias ARG, Figueira AR, Geraldino PS, Pompeu DC. First Report of the Occurrence of Banana streak Goldfinger Virus (BSGFV) in the State of Minas Gerais, Brazil. Adv Biotech & Micro. 2018; 10(5): 555799. DOI:10.19080/AIBM.2018.10.555799
Abstract
The leaf streakviral disease inbanana plants iscaused by a species complex known as Banana streak virus(BSV). In Brazil, there have been few studies concerningthese species, Butcharacterizing them is necessary to determine which species are endemicin the field. To this end, banana leaf samples were collected from different producing regions of Brazil. Their total extracted DNA was then subjected to Polymerase Chain Reaction (PCR)employing theBadna-FP/-RP primers to amplify a 540-base-pair (bp) fragment located in the gene that encodesRT/Rnase H, which is usedto differentiate Badnavirus species.The positive samples were confirmed by Rolling Circle Amplification (RCA) using the Illustra TempliPhi 100 Amplification Kit. The 540-bp genome fragment wassequenced and the results analyzed and compared with the Badnavirus species available inthe GenBank database. The nucleotide sequence of the isolate designated MGJAPI, when compared to the GenBank BSGFV isolates, showed the lowest identity percentage,91%, with the Uganda isolate (AJ968435). When compared to other isolates, the identities ranged from 97% to 99%.Thus, according to the International Committee on the Taxonomy of Viruses (ICTV) classification criteria, the isolate MGJAPI belongs to the species Banana streak Goldfinger virus (BSGFV) and this is the first reportof the occurrence of this species in the state of Minas Gerais, Brazil..
Keywords:leaf streakviral disease; plants; Badnavirus species; Goldfinger virus; seedlings; Cucumber mosaic virus; Uganda virus; Planococcus citriand; Saccharicoccus sacchari; banana; Vietnam virus; Climate; pathogens; Brazilian farmers; Amino acid; Percentage; Amino acid; Cladograms; Crop; Fruit
Abbrevations:BVS: Banana streak virus; PCR: Polymerase Chain Reaction; RCA: Rolling Circle Amplification; ICTV: International Committee on Taxonomy of Viruses; UPGMA: Unweighted Pair Group Method with Arithmetic
Introduction
Brazil is the fourth-largest producer of bananas in the world, cultivatingabout seven million tons overan area of approximately 500,000 hectares.The favorable Brazilian climate enables the productionof fruit year-round (FAO, 2012). As favorable as the climate is for producing bananas, it is also favorable forthe development of several pathogens, which increasethe cost of production and constitute a serious challenge for Brazilian farmers. The propagation of healthy seedlings has been one of the most important phytosanitary measuresfor controlling these diseases, especially when it comes to viral diseases [1]. This is because once infected,plants cannot be cured and must be eliminated from the crop, causing undesirable losses.
There are two viral diseasesfound in the banana tree in Brazil:infectious chlorosis of banana, caused by the Cucumber mosaic virus (CMV), and the other is banana leaf streak, caused by the Badnavirus species [2]. However, the process of indexing seedlings to a diagnosis of Badnavirus is quite complicated, since in addition to presenting high genetic variability, Badnavirus species associated with Banana streak are able to integratesome or all of their DNA into the banana plant’s genome, compromising the efficiency of serological and molecular testing techniques [3-6].
In addition to the Banana streak virus (BSV), other species of Badnavirus associated with Banana streak, such as Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV) have already been well characterized [7,8]. However, as the genomes of the different isolates that have been found are sequenced, new species appear, such as Banana streak acuminata Vietnam virus (BSAcVNV) [9], Banana streak Cavendish virus (BSCavV) [10], Banana streak Imové virus (BSImV),Banana streak Uganda A-M virus(BSUgBV) [11,8], and many others. This has hampered the taxonomic classification of this virus, and sometimes the isolates are identified asBSV species but, in fact, the BSV was only the first species of the genus Badnavirus described in banana. The main vectors that disseminate these species in the field are the mealybugs Planococcus citriand Saccharicoccus sacchari, but it has not been ruled out that they can also be transmitted by other species of mealybug [12, 13].
The PLs are diverse in the site of action on phospholipids molecules and therefore they are classified into 4 types namely A, B, C and D (Figure 1) (Table 1). Phospholipase A (PLA) is further classified into two subtypes A1 that cleave the acyl ester bond at sn- 1 position and A2 cleaves at sn- 2 position. On the bases of cellular localization the isozymes of PLA1 is divided into two groups i.e. intracellular and extracellular enzymes (Figure 2) [16]. Similarly, PLA2 has been sorted into 5 main types that further contain different groups (group I-XV) (Figure 3) [17]. Some PLs hydrolyze both acyl groups and are termed the phospholipases B (PLB), also known as lysophospholiapse [18]. Enzymes grouped under phospholipase C (PLC) cleaves glycerophosphate bond on the glycerol side, while phospholipase D (PLD) catalyses the removal of base group on the polar side of phospholiapse [19,20]. The PLC and PLD are therefore also known as phosphodiesterases. Till now, 13 PLC isoenzymes have been identified that are grouped into six different subfamilies (Figure 4) [20]. There are two isoforms of PLD (Figure 5). In addition to bacteria, PLD has been reported in many plants, viruses, worms, flies and yeast [21].
Structural analysis
It is very important to understand the three dimensional structure of enzyme to examine its significant role in the pathophysiology of the microorganisms in disease. PLA1 contains the lipase consensus sequence (Gly-x-Ser-x-Gly), a catalytic triad (Ser-Asp-His) with serine at its active site and shares a multiple conserved motifs. Intracellular PLA1 mainly contains DDHD domain and some of them have a sterile alfa motif (SAM) which is important for the binding of enzyme to the intracellular membrane [22]. Extracellular PLA1 contains surface loops known as lids, β5 loops and β9 loops. Subfamilies present in secretary PLA1 mainly varies due to the length of lids and β9 loops. Notably, PLA1 that posses triacylglycerol hydrolase activity generally have long lids (22-23 amino acids) and long β9 loops (18-19 amino acids), whereas PLA1-α and PLA1- β that do not exhibits triacylglycerol hydrolase activity contain short lids (7-12 amino acids) and β9 loops (12-13 amino acids) [23].
PLA2 belongs to the α/β hydrolse family. β- sheets are generally present in the enzyme core whose strands are interconnected by α- helices and catalytic serine is present in tight turn between α/β strand. Secreted PLA2 contains His-Asp catalytic diad and a Ca2+ binding site, whereas cytosolic PLA2 contains Ca2+ dependent phospholipid domain and its active site is covered by the cap region [24]. Plasma platelet activating factor-acetylhydrolase and Lysosomal PLA2 contains lipase motif (Gly-x-Ser-x-Gly), the catalytic Ser-Asp-His triad, and serine active site. They also contains the N-glycolation site and an N-terminal signal sequence [25].
PLC contains X and Y catalytic domain that comprise highly conserved amino acid regions in isozymes. They are located between EF-hand motif which is a helix-turn-helix structural domain that binds Ca2+ ions and C2 domain that also contains three to four Ca2+ binding sites and regulates the enzyme activity. It has been shown that plekstrin homology (PH) domain is located in the N- terminal region and it provides the passage onto the membrane surface [20]. PLC- β and PLC-γ contains an additional COOH- terminal and SH domain respectively that is responsible for the membrane attachment and in mitogenic signaling [20]. PLD contains the sequence motif HXK(X)4D, which is found twice without exception in all known isoforms of enzyme and denoted as HKD motif [21]. This motif has been involved in signal transduction and lipid biosynthesis in many pathogenic bacteria [26]. Phox consensus sequence (PX), the (PH) domain is the other highly conserved regions which are involved in lipid binding [27].
Mechanism of action
The PLA gene family member shares a multiple conserved motif that includes G-X-S-X-G motif, a catalytic triad and cysteine residues that moderated disulphide bond formation. It contains the N-terminal signal sequence followed by catalytic triad with Ser154, Asp178, and His249. Glycosylation is critical for the catalytic activity and four acceptors sequences of N-glycosylation sites are present at amino acid 50, 58, 66 and 357 positions [28]. The catalytic action of PLA2 proceeds through the Serine–acyl intermediate that is present in a pentapeptise sequence G-L-LG- S using serine-228 as nuclephilic residue. This catalytic serine residue is termed as “nucleophilic elbow”. It has been reported that in addition to Serine-288, Asp-549 and Arg-200 is also found to be essential for the activity [24].
The X and Y structural domain of PLC are responsible for the catalytic activity of the enzyme. Based on the structural analysis it has been reported that Lysine-438, Lysine-440, Serine-522 and Arginine-549 are present at active site which are implicated in the binding with the phosphate group [20]. PH domain moderates the binding of enzyme to phospholipids. PLC-γ contains the long amino acid sequence that contain Src homology (SG) domain which mediated the interaction with other proteins [29]. Phospholipase D crystal structure reveals that it contain two motif from single active site and the histidine residue from one motif acts as a nucleophile in the catalytic mechanism of the enzyme forming an intermediate of phosphoenzyme whereas, the histidine residue of second motif cleaves the phosphodiester bond [26]. PH and PX domain also plays an important role in the catalysis of enzyme. PH domain helps in the localization of the protein whereas; PX domain is thought to mediate the protein- protein interaction. The lysine residue conserved in the structure is involved in phosphate binding [27].
Role in virulence and pathogenesis: Several bacteria and fungi produce extracellular phospholipases, which helps them to invade the host by damaging its cell membrane [8,30,31]. The presence of their activity is generally associated to the virulence of the pathogen. The strains of Candida albicans with highest phospholipase activity showed the greatest mortality in mice [8]. Also, only phospholipase activity was predictive of mortality among a number of candidate factors in C. albicans [32]. It has been reported that Aspergillus fumigatus was able to produce different type of phospholipases like PLA, PLB, PLC, and PLD [33]. Different phospholipases that play an important role in bacterial virulence and pathogenesis are mentioned in Table 2.
Phospholipases are important virulence factors as they are able to cleave phospholipids in eukaryotic membranes and the products might act as signaling molecules, which ultimately leads to a number of events to occur favorable for the pathogen [14]. They help the bacterial pathogens to invade the host cells by destroying the phospholipids of cell membranes. The role of α-toxin (PLC) was confirmed when α-toxin mutant from a virulent strain of Clostridium perfringens was unable to cause tissue damage and necrosis in mice hind limbs after inoculation [34]. Also, they modulates phospholipid content of cell envelope of certain bacteria, which would be helpful for the pathogenesis of bacteria like PLA1 of Brucella melitensis is responsible for the resistance against polymyxin B and also contributed to host-pathogen interactions [35]. There are three PLDs in Acinetobacter baumannii and are major virulence factors as they are required for host cell invasion. All three PLDs were necessary for the full invasion and virulence as they work in concerted manner, confirmed when the inactivation of all three pld genes leads to the minimum invasion efficiency [36]. Moreover, they play an important role in pathogenesis of intracellular pathogens as they help the bacteria to escape from phagosomes in certain cases. Two PLC were found in Listeria monocytogenes which aid the bacterial escape from phagosomes as this was confirmed by creating its mutants. The individual mutants of plcA or plcB were two and 20 fold, respectively, less virulent, but a double mutant was 500-fold less virulent in mice deciphering the significance of this enzyme for the virulence and pathogenesis of the bacteria [37].
The intracellular lung pathogen, Mycobacterium tuberculosis also possessed phospholipases which are important for its virulence and pathogenesis too. There are four genes (plcA, plcB, plcC, plcD) that encode PLC enzyme. Mutation studies demonstrated that all four genes were required to encode a functional PLC. The expression of these genes was upregulated during first 24 hr of infection suggesting the role of PLC in the virulence of the bacteria [38].
Sometimes, they are very crucial to the pathogens that without them bacteria would be unable to survive in the host. One such example is the PLA2 enzyme of Streptococcus pneumoniae which elicits pulmonary inflammation during infection and is also required for lethal systemic infection [39]. PLA2 enzyme inhibitors almost blocked (diminished by >80%) the polymorphonuclear cells (PMN) transepithelial migration in vitro [40]. Also, PLA2-deficient mice were survived from S. pneumoniae bacteremia challenge which was otherwise lethal to wild-type mice [41]. The byproducts of this enzyme catalysis lead to the formation of certain metabolites that aid in the inflammatory processes and in that case phospholipase inhibition could be a more effective anti-inflammatory approach [42]. Like bacteria, snake venom is enormously rich in these enzymes and their inhibitors could prevent skeltel muscle necrosis and permanent injuries in snakebite victims [43].
They also play anabolic roles. There are two PLA2 that are pivotal in lipid droplet formation in case of Hepatitis C virus infection (HCV). Their knockdown studies showed that their function were irreplaceable and could not be restored even on complementation with each other and lipid droplet formation activity was also found to be impaired. These two PLA2 were found to be play an important role in HCV replication and pathogenesis and they could be a target for an anti-HCV drug [44-68].
Materials and Methods
Virus isolates: origin and maintenance
Samples of banana plants suspected of being infected with BSV were collected in different regions of the countryduring visits to fields, tissue culture laboratories, and with the collaboration of professionals working in banana production whosent them by mail or other appropriate transport.
PCR, sequencing, sequence analysis
After extracting the total DNA from samples of the potentiallyinfected leaves, the remaining leaves were stored in a freezer at -80 °C anddesiccated for future use. Samples of bananaleaves that were cultivated in a controlled greenhouse environmentat the Federal University of Lavras (UFLA), and which proved to be virus-free, were used as negative controls.
Total DNA from the healthy and infected leaves was extracted, following the manufacturer’s instructions for the Extract-N-Amp Plant PCR kit (Sigma Aldrich)and stored at -80 °C for later use. The DNA samples were submitted to PCR (polymerase chain reaction) and the positive samples were again analyzed by RCA (rolling circle amplification)following the methodology described by James et al. [6], to verify if the amplified sequences were episomal. PCR was performed using the primers Badna FP and Badna RP [17], which amplify a fragment of 540 base pairs, corresponding to part of the encoding region RT/RnaseH of Badnavirus.After confirmation of DNA amplification by conventional PCR, the samples were submitted to RCA, following the recommendations of the manufacturer’s manualfor the Illustra TempliPhi 100 Amplification Kit (GE Healthcare, Buckingamshire, United Kingdom).The samples’ DNA (1μl) was added to 5μl of buffer and 1μl of each primer at the concentration of 60μM. The mixture was incubated at 95°Cfor 3 minutes and then 5μl of reaction buffer,previously mixed with 0.2 μlof phi29 DNA polymerase,was added. The reaction was incubated at 30 °C for 12 hours and stopped with incubation at 65°C for 10 minutes.
The genomic fragments of 540 base pairswere sequenced by the company Myleus Biotecnologia and the analyses were carried out using the program NCBI BLAST () [18]. Alignment of the nucleotide and amino acid sequences and the construction of phylogenetic trees was performed using the programs ClustalW2 (Verson 2.0) and Molecular Evolutionary Genetics Analysis - MEGA6 [19], respectively. The characterization of the species followed the criteria of the International Committee on Taxonomy of Viruses (ICTV), which considers a species of Badnavirus sp. as distinct from others by having an identity difference above 20% in the RT/RnaseH coding region.The phylogenetic relationships were studied using the algorithm neighbor- joining for amino acids andthe Unweighted Pair Group Method with Arithmetic Mean (UPGMA)for nucleotides, with 3,000 bootstraprepetitions.
Results
Comparing the nucleotides of the isolate designated as MGJAPI with the BSGFV isolatesin the GenBank database shows that its lowest identity percentage was 91%.With isolate AJ968435 from Uganda and with the other isolates used for comparison, it was above 97%, with a maximum of 99% identity.The lower identity percentage (95%) forthe amino acid sequence was also observed with the Uganda isolate already cited, and with the other isolates the identity was 98%.Based on the ICTV criteria, which considers particular isolates to be members of the same species if they share identities above 80%in the genomic fragment that corresponds to the RT/RNase H, the isolate MGJAPI was identified as BSGFV.
The cladograms based on the sequence of nucleotides and amino acids showed a clear grouping of MGJAPI and BSGFV species, confirming the identity results previously shown.This is the first time that the species BSGFV has beendetected and characterized in Brazil [20].
Conclusion
There is a wide variety of the Banana streak virusspecies in Brazil.However, Banana streak Goldfinger virus has been identified for the first timein the state of Minas Gerais.New samples are being collected and analyzed to obtain better knowledge of the Badnavirus species present in Brazil.
Acknowledgement
The authors are grateful to the producers and researchers committed to banana production in Brazil who provided samples for study, and also the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), andthe Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the resource funds to support their current work.
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