Sugarcane Yellow Leaf Virus Isolates Research Articles

Genetic Diversity, Evolution, and Diagnosis of Sugarcane Yellow Leaf Virus from 19 Sugarcane-Producing Locations Worldwide.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf, has been reported in an increasing number of sugarcane-growing locations since its first report in the 1990s in Brazil, Florida, and Hawaii. In this study, the genetic diversity of SCYLV was investigated using the genome coding sequence (5,561 to 5,612 nt) of 109 virus isolates from 19 geographical locations, including 65 new isolates from 16 geographical regions worldwide. These isolates were distributed in three major phylogenetic lineages (BRA, CUB, and REU), except for one isolate from Guatemala. Twenty-two recombination events were identified among the 109 isolates of SCYLV, thus confirming that recombination was a significant driving force in the genetic diversity and evolution of this virus. No temporal signal was found in the genomic sequence dataset, most likely because of the short temporal window of the 109 SCYLV isolates (1998 to 2020). Among 27 primers reported in the literature for the detection of the virus by RT-PCR, none matched 100% with all 109 SCYLV sequences, suggesting that the use of some primer pairs may not result in the detection of all virus isolates. Primers YLS111/YLS462, which were the first primer pair used by numerous research organizations to detect the virus by RT-PCR, failed to detect isolates belonging to the CUB lineage. In contrast, primer pair ScYLVf1/ScYLVr1 efficiently detected isolates of all three lineages. Continuous pursuit of knowledge of SCYLV genetic variability is therefore critical for effective diagnosis of yellow leaf, especially in virus-infected and mainly asymptomatic sugarcane plants.

Comparative genomics reveals insights into genetic variability and molecular evolution among sugarcane yellow leaf virus populations

Yellow leaf disease caused by sugarcane yellow leaf virus (SCYLV) is one of the most prevalent diseases worldwide. In this study, six near-complete genome sequences of SCYLV were determined to be 5775–5881 bp in length. Phylogenetic analysis revealed that the two SCYLV isolates from Réunion Island, France, and four from China were clustered into REU and CUB genotypes, respectively, based on 50 genomic sequences (this study = 6, GenBank = 44). Meanwhile, all 50 isolates were clustered into three phylogroups (G1–G3). Twelve significant recombinant events occurred in intra- and inter-phylogroups between geographical origins and host crops. Most recombinant hotspots were distributed in coat protein read-through protein (RTD), followed by ORF0 (P0) and ORF1 (P1). High genetic divergences of 12.4% for genomic sequences and 6.0–24.9% for individual genes were determined at nucleotide levels. The highest nucleotide diversity (π) was found in P0, followed by P1 and RdRP. In addition, purifying selection was a main factor restricting variability in SCYLV populations. Infrequent gene flow between Africa and the two subpopulations (Asia and America) were found, whereas frequent gene flow between Asia and America subpopulations was observed. Taken together, our findings facilitate understanding of genetic diversity and evolutionary dynamics of SCYLV.

A survey of causal agents associated with sugarcane yellowing in Northeast Brazil

The etiological agents of the yellow leaf syndrome (YLS) are the Sugarcane yellow leaf virus (ScYLV), and phytoplasmas of different groups. Due to a lack of knowledge on the occurrence of YLS and the importance of sugarcane to Brazil, the aims of this study were to assess the occurrence and distribution of the pathogens associated with YLS in sugarcane cultivation areas in three states of Northeast Brazil. For this purpose, 101 sugarcane samples with symptoms of YLS were collected in 2018 crop cycle. ScYLV and a new phytoplasma were detected by RT-PCR and nested PCR, respectively. The identity of virus- and phytoplasma-positive samples was confirmed by Sanger sequencing, and these sequences were used in subsequent phylogenetic studies. Both pathogens were present in all sampled regions. ScYLV and the phytoplasma were detected in 40.4% and 62.2% of the samples, respectively. Mixed infections were found in 33.5% of the samples. Phylogenetic analysis showed that the ScYLV isolates identified were most closely related to ScYLV genotypes REU, PER, HAW, CHN2 and BRA. The phytoplasma 16S rDNA gene nucleotide sequences shared 99.0% identity with sequences of the 16SrVII phytoplasmas from Argentina and Brazil. This is the first report of the presence of a member of the 16SrVII phytoplasma group in association with sugarcane leaf yellowing in the world. These findings are important to raise awareness among sugarcane producers on the impact of pathogens causing YLS in Northeast Brazil.

Population structure and selection pressure analysis among Sugarcane yellow leaf virus isolates based on P0 and P1 sequences

Yellow leaf of sugarcane is caused by Sugarcane yellow leaf virus (SCYLV) and has been a threat to global sugar industry. Understanding the diversity and genetic structure of plant virus populations may help to develop strategies to control virus-induced diseases. In this study, demography, genetic differentiation and selection pressure were assessed based on 57 nucleotide sequences of the P0 (567 nt) and 54 nucleotide sequences of the P1 (1413 nt) genes of SCYLV isolates. Phylogenetic analysis revealed three phylogeographical groups: African (Clade I), Chinese/American (Clade II), and Asian (Clade III). Genetic distance values between groups (0.034–0.250) were higher than within-group values (0.011–0.033). The African group was in a state of expansion, with lower nucleotide sequence diversity. Except for those isolates within the Asian group, SCYLV showed infrequent gene flow within or between phylogenetic groups. Analysis of selection pressure suggested that these two genes have been subjected to negative (purifying) selection. However, some sites, especially in the P0 coding region of the Chinese/American group, have undergone positive selection. Overall, aside from recombination and founder effects, gene flow and purifying selection are two important factors driving SCYLV evolution.

Genotyping of Sugarcane Yellow Leaf Virus in Commercial Cultivars and the Cuban Germplasm Collection

Sugarcane yellow leaf disease caused by Sugarcane yellow leaf virus (SCYLV) was spread over the world through infected germplasm. This study was conducted in order to determine the presence of SCYLV in commercial fields and germplasm collection and study the genetic diversity of the virus in Cuba. Leaf samples were collected from commercial cultivars in eight provinces and the Cuban germplasm collection in Matanzas province. The diagnosis of SCYLV was performed using tissue-blot immunoassay and reverse transcription polymerase chain reaction (RT-PCR). Our results revealed the widespread incidence of SCYLV in commercial sugarcane plantations in Cuba. The coat protein sequence analyses of SCYLV isolates under study confirmed the presence of BRA-PER, CUB and REU genotypes. The REU genotype is reported first time in Cuba. Coinfections of SCYLV genotypes (BRA-PER + CUB, BRA-PER + REU and CUB + REU) were also reported. The most predominating SCYLV genotypes reported in Cuba were identified as CUB and BRA-PER.

Prevalence of Sugarcane yellow leaf virus in Sugarcane-Producing Regions in Kenya Revealed by Reverse-Transcription Loop-Mediated Isothermal Amplification Method

Yellow leaf (YL) is a disease of sugarcane that is currently widespread throughout most American and Asian sugarcane-producing countries. However, its actual distribution in Africa remains largely unknown. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to facilitate and improve the detection of Sugarcane yellow leaf virus (SCYLV), the causal agent of YL. The RT-LAMP assay was found to be comparable with or superior to conventional RT-polymerase chain reaction (PCR) for the detection of SCYLV genotypes CUB and BRA in infected sugarcane 'C132-81' and 'SP71-6163', respectively. Additionally, 68 sugarcane samples that tested negative by RT-PCR were found positive by RT-LAMP, whereas the RT-LAMP assay failed to detect SCYLV in only 5 samples that tested positive by RT-PCR. Combining RT-PCR and RT-LAMP data enabled the detection of SCYLV in 86 of 183 Kenyan sugarcane plants, indicating high SCYLV prevalence throughout the country (ranging from 36 to 64% in individual counties). Seminested PCR assays were developed that enabled the amplification of a fragment encompassing the capsid protein coding region gene and its flanking 5' and 3' genomic regions. Sequences of this fragment for four Kenyan SCYLV isolates indicated that they shared 99.2 to 99.6% pairwise identity with one another and clearly clustered phylogenetically with SCYLV-BRA genotype isolates. To our knowledge, this is the first report of SCYLV in Kenya.

Molecular detection, genetic diversity and distribution of Sugarcane yellow leaf virus genotypes in China

A total of 332 sugarcane leaf samples were collected from seven provinces in China during 2010 to 2013. RT-PCR was performed using the YLS111/YLS462 primers to detect the causal pathogen Sugarcane yellow leaf virus (SCYLV) causing yellow leaf disease in sugarcane. The incidence of SCYLV infection ranged from 24 to 38 % depending on geographic areas. A new primer pair (P0-R3/P0-F2) was designed to prime the amplification of a 968-nucleotide (nt) fragment from SCYLV-positive samples. The fragment contained a partial 5′ untranslated region, the complete ORF0 encoding P0, and a partial ORF1. A phylogenetic analysis of 141 P0 fragment sequences (937 nt) worldwide showed that all SCYLV isolates were clustered into eight genotypes. The 107 SCYLV isolates collected in this study were classified into three genotypes (BRA, HAW, and CHN3), of which the BRA genotype (102/107) was the most prevalent and was detected in all evaluated locations. One isolate (1/107) of genotype HAW was found in Hainan province. Four isolates (4/107) of the genotype CHN3 were observed in Guangxi, Guizhou and Hainan provinces. These findings help understand the genotypic diversity and distribution of SCYLV isolates in China.

First Report of Sugarcane yellow leaf virus Infecting Barley in Tunisia.

Sugarcane yellow leaf virus (ScYLV) causes severe leaf symptoms in sugarcane (Saccharum spp.). It is a single-stranded RNA virus assigned to the genus Polerovirus, family Luteoviridae (1). ScYLV is transmitted by two aphid species, Melanaphis sacchari and Rhopalosiphum maidis. Although barley (Hordeum vulgare), oats (Avena sativa), and wheat (Triticum spp.) are susceptible to ScYLV when experimentally inoculated (3), this virus, related serologically to Barley yellow dwarf virus (BYDV)-RPV (4), has never been detected naturally in these cereals. In this study, 240 barley leaves were randomly collected from six fields in Tunisia following a north-south trend during the high infestation periods (March/April) in the 2013 growing season. Samples were tested by DAS-ELISA, using three antibodies (Bioreba AG, Switzerland), two of them, BYDV-B and BYDV-F, specific to luteoviruses corresponding to BYDV-PAV and BYDV-MAV, respectively, and the third one, BYDV-RPV, specific to the polerovirus synonymous to Cereal yellow dwarf virus (CYDV)-RPV. Based on DAS-ELISA, 30 samples were found positive for B/CYDV infection; 17 out of the 30 infected samples contained a single serotype, BYDV-PAV, and 13 out of the 30 infected samples contained two serotypes, PAV and RPV. Total RNA was extracted from all positive samples, and RT-PCR of the viral CP gene was performed with Lu1/Lu4 primers (2). A product of 531 bp was cloned and sequenced. The identities among the sequences determined varied between 80 to 100%, and from the 17 samples containing BYDV-PAV, six distinct BYDV-PAV sequences were revealed and named PAV-TN1 to PAV-TN6 (GenBank Accession No. JX402453 to JX402457 and KF271792). Fortuitously, all 13 positive samples corresponding to the serotypes PAV-RPV exhibited 98.7 to 99.3% identity with ScYLV isolates. These 13 samples contained three distinct sequences that were named ScYLV-Tun1 to ScYLV-Tun3 (GenBank Accession No. KF836888 to KF836890). Of the 17 PAV-positive samples collected, six were infected with PAV-TN1, four with PAV-TN2, four with PAV-TN3, one with PAV-TN4, one with PAV-TN5, and the last one with PAV-TN6. Of the 13 ScYLV-positive samples, seven were infected with ScYLV-Tun1, four with ScYLV-Tun2, and two with ScYLV-Tun3. Phylogenetic analysis showed that PAV-TN sequences formed a very tight cluster (>98%) corresponding to BYDV subspecies PAV-II, whereas all three Tunisian ScYLV sequences were clustered together. This study provides the first report of ScYLV isolates infecting barley crops in Tunisia, and confirms serological cross-reactivity between ScYLV and BYDV-RPV when commercial antibodies against BYDV-RPV are used.

Molecular evolutionary history of Sugarcane yellow leaf virus based on sequence analysis of RNA-dependent RNA polymerase and putative aphid transmission factor-coding genes.

RNA-dependent RNA polymerase (RdRp) encoded by ORF2 and putative aphid transmission factor (PATF) encoded by ORF5 of Sugarcane yellow leaf virus (SCYLV) were detected in six sugarcane cultivars affected by yellow leaf using RT-PCR and real-time RT-PCR assays. Expression of both genes varied among infected plants, but overall expression of RdRp was higher than expression of PATF. Cultivar H87-4094 from Hawaii yielded the highest transcript levels of RdRp, whereas cultivar C1051-73 from Cuba exhibited the lowest levels. Sequence comparisons among 25 SCYLV isolates from various geographical locations revealed an amino acid similarity of 72.1-99.4 and 84.7-99.8% for the RdRp and PATF genes, respectively. The 25 SCYLV isolates were separated into three (RdRp) and two (PATF) phylogenetic groups using the MEGA6 program that does not account for genetic recombination. However, the SCYLV genome contained potential recombination signals in the RdRp and PATF coding genes based on the GARD genetic algorithm. Use of this later program resulted in the reconstruction of phylogenies on the left as well as on the right sides of the putative recombination breaking points, and the 25 SCYLV isolates were distributed into three distinct phylogenetic groups based on either RdRp or PATF sequences. As a result, recombination reshuffled the affiliation of the accessions to the different clusters. Analysis of selection pressures exerted on RdRp and PATF encoded proteins revealed that ORF 2 and ORF 5 underwent predominantly purifying selection. However, a few sites were also under positive selection as assessed by various models such as FEL, IFEL, REL, FUBAR, MEME, GA-Branch, and PRIME.

Complete genome characterization of Sugarcane yellow leaf virus from India: Evidence for RNA recombination

Yellow leaf (YL) caused by Sugarcane yellow leaf virus (SCYLV) has become a serious constraint for sugarcane production in different countries. Worldwide seven genotypes have been reported, with five based on complete and two based on partial genome characterization. We have previously reported the occurrence of three different SCYLV genotypes in India based on their partial genome sequences. A further four SCYLV isolates from sugarcane from Coimbatore (in India) were characterized after complete genome sequencing (~ 5,875 nt). These isolates (SCYLV-IND) exhibited amino acid (aa) sequence differences of 29.2–31.8, 28.1–34.4 and 30.7–33.4 % with REU, HAW-PER and BRA in partial ORF0 sequences, respectively. Similarly IND isolates have 21.4–23.7, 22.5–25.0 and 21.4–23.9 % aa sequence differences with REU, HAW-PER and BRA, respectively in partial ORF1. However, the difference was found to be least in ORF5. The genotype reported from China, CHN1 shared a very close relationship with IND isolates with minimum differences of 4.3–5.3 %, 4.8–5.8 % and 2.5–3.0 % in ORF0, 1 and 5 in aa sequences, respectively and 4.4–5.3 % in complete nucleotide sequences. Phylogenetic analyses showed a separate lineage for IND isolates. Evidence of recombination was found in ORF1 to ORF5 with the maximum number of sites occurring in ORF2. The high incidence of SCYLV recombination suggests that recombination plays an important role in SCYLV evolution.

Molecular characterization and phylogenetic analysis of Sugarcane yellow leaf virus isolates from China

Sugarcane yellow leaf virus (SCYLV) (genus Polerovirus, family Luteoviridae), the causal agent of sugarcane yellow leaf disease (YLD), was first detected in China in 2006. To assess the distribution of SCYLV in the major sugarcane-growing Chinese provinces, leaf samples from 22 sugarcane clones (Saccharum spp. hybrid) showing YLD symptoms were collected and analyzed for infection by the virus using reverse transcription PCR (RT-PCR), quantitative RT-PCR, and immunological assays. A complete genomic sequence (5,879 nt) of the Chinese SCYLV isolate CHN-FJ1 and partial genomic sequences (2,915 nt) of 13 other Chinese SCYLV isolates from this study were amplified, cloned, and sequenced. The genomic sequence of the CHN-FJ1 isolate was found to share a high identity (98.4-99.1 %) with those of the Brazilian (BRA) genotype isolates and a low identity (86.5-86.9 %) with those of the CHN1 and Cuban (CUB) genotype isolates. The genetic diversity of these 14 Chinese SCYLV isolates was assessed along with that of 29 SCYLV isolates of worldwide origin reported in the GenBank database, based on the full or partial genomic sequence. Phylogenetic analysis demonstrated that all the 14 Chinese SCYLV isolates clustered into one large group with the BRA genotype and 12 other reported SCYLV isolates. In addition, five reported Chinese SCYLV isolates were grouped with the Peruvian (PER), CHN1 and CUB genotypes. We therefore speculated that at least four SCYLV genotypes, BRA, PER, CHN1, and CUB, are associated with YLD in China. Interestingly, a 39-nt deletion was detected in the sequence of the CHN-GD3 isolate, in the middle of the ORF1 region adjacent to the overlap between ORF1 and ORF2. This location is known to be one of the recombination breakpoints in the Luteoviridae family.

Investigation of ORF0 as a sensitive alternative diagnostic segment to detect Sugarcane yellow leaf virus

The worldwide distribution of Sugarcane yellow leaf virus (SCYLV) has led several research groups to study the function of the viral genome, the role of open reading frames (ORFs), their influence on virus accumulation and methods for diagnosis. The detection of SCYLV is usually based on the viral coat protein whether using serological and/or molecular techniques. In this study, ORF0 has been used as a diagnostic segment for SCYLV due to its highly conserved region in all SCYLV isolates. The results revealed that, ORF0 was expressed more consistently in all cultivars. In contrast, the expression of the coat protein varied. The RNA poleroviruses sequences of ORF0 were variable compared with the same segment of SCYLV populations. Analysis of the amino acid sequence of the ORF0 translation product revealed the presence of a potential transmembrane domain. The relatively high content of hydrophobic amino acids in the ORF0 protein further suggests that it may serve as a membrane anchor for the replication complex.

Putative recombination signature and significance of insertion/deletion events in the RNA-dependent RNA polymerase coding region of sugarcane yellow leaf virus

The 5898 nucleotide single-strand RNA genome of Sugarcane yellow leaf virus (SCYLV) contains one long open reading frame, which is translated into a 120.6kDa polyprotein. The sequences of SCYLV isolates from the two SCYLV-susceptible cultivars from Hawaii had a deletion of 48-51nt in ORF1. SCYLV from 12 sugarcane hybrid cultivars from different origins were tested by RT-PCR using a specific set of primers, to investigate the genome segment for this deletion. Only three cultivars were found not to have the deletion (H87-4319, JA-605 and CP52-43), while SCYLV from nine cultivars (H73-6110, H87-4094, H78-7750, GT54-9, G84-47, H78-4153, H65-7052, C1051-73, Ph-8013) along with aphid (Melanaphis sacchari), which fed on SCYLV-infected H73-6110, contained a deletion of about 50nt. The deleted sequence was located in the overlap frameshift of ORF1 and ORF2. Thus, ORFs 1 and 2 of SCYLV are translated via ribosomal frameshift and yield the 120.6kDa viral replicase. ORF1 plays most likely a role in the replication and is a source of large variability among the virus population. To identify possible recombination events located in the RdRp domain of the Hawaiian isolates, two programs were used: RDP v.4.3 and RECCO. It is noteworthy that according both methods Haw73-6110 was found as a potential recombinant. On the other hand, opposed to the RDP package, RECCO revealed that Haw87-4094 isolate was also a recombinant whereas Haw87-4319 was not.

Genotype identification and genetic diversity of Sugarcane yellow leaf virus in China

A survey of Sugarcane yellow leaf virus (SCYLV) in China showed infection rates of 1·1–42·4% in six different sugarcane growing provinces, with an average rate of 20·3% (129/634), and no infection in one province. The majority of the infected plants showed yellowing of the midrib on the abaxial surface of leaves. Genotypic diversity of the 129 SCYLV isolates was analysed by a reverse transcription–polymerase chain reaction–restriction fragment length polymorphism (RT‐PCR‐RFLP) assay and nucleotide sequencing. Sixty‐six isolates from Guangxi, Yunnan, Hainan, Fujian and Guizhou provinces were of the genotype BRA, whereas another 63 isolates from Guangdong province consisted of five genotypes, BRA, CHN1, CUB, PER and a novel genotype, CHN2. Among these genotypes, BRA was the predominant one identified in 43 samples (68·3%). Mixed infection of BRA and PER was also found in two samples. Phylogenetic analysis based on a 1·3‐kb nucleotide sequence (including ORFs 3–4 and partial ORF 5) supported the identification of the novel CHN2 genotype. CHN2 shared 91·9–97·8% identity with five other known genotypes and was most closely related to the CHN1 genotype (97·6–97·8% identities). The nearly complete genomic sequences of seven isolates representing genotypes BRA, CUB and PER were determined, and they shared nucleotide sequence identities of 97·2–97·5%, 98·4–98·9% and 98·4%, respectively, with previously reported sequences of the corresponding genotypes.

Molecular detection and identification of thirteen isolates of Sugarcane yellow leaf virus associated with sugarcane yellow leaf disease in nine sugarcane growing states of India

Thirty sugarcane leaf samples exhibiting midrib yellowing symptoms from nine sugarcane growing states of India were collected. The total RNA was isolated from infected samples and RT-PCR assays were performed using Sugarcane yellow leaf virus (SCYLV) specific primers. The infection of SCYLV was detected in 27 out of 30 samples, which showed the expected size (~610 bp) amplicon during RT-PCR. The amplicons from 13 samples were cloned, sequenced and sequence data of these isolates were analyzed to determine sequence identities, sequence variations and phylogenetic relationships with the SCYLV isolates reported earlier from India and abroad with the view to identify the virus isolates based on partial sequence of ORF5, and complete ORF3 (coat protein, CP) and ORF4 (movement protein, MP). The analysis of the complete sequence data (613 nucleotides) of 13 isolates under study revealed 96–100% identities among them and also with SCYLV-Cuban isolate. The identities were 90–94% with other isolates of SCYLV reported from other countries. The nucleotide (nt) and amino acid (aa) sequence comparison in the CP and MP coding regions showed a significant variation between Indian isolates (SCYLV-IND) and other SCYLV isolates reported worldwide. Phylogenetic analyses of the 13 SCYLV isolates under study showed that they clustered together along with most other SCYLV-Indian isolates and a SCYLV-Cuba isolate. The remaining isolates from other regions of the world and a single isolate from India characterized in a previous study clustered into separate groups. Therefore, the isolates under study were identified as isolates of SCYLV which are closely related to SCYLV-Cuba, the member of ‘CUB’ genotype.

Molecular detection of two strains ofSugarcane yellow leaf virusin India and their secondary spread in nature through aphids

Thirteen Sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot patterns from six states of India were characterized by RT-PCR assays. SCYLV-615F and SCYLV-615R primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. Comparative coat protein sequence analysis confirmed that all the SCYLV-Indian isolates were clustered into two major groups confirming the existence of two strains of SCYLV affecting sugarcane crops of India. However, all the 13 isolates under study were found related with Cuban isolate and belong to the CUB genotype. We concluded that YLD in India is caused by two genotypes CUB and IND, rather than by three genotypes as reported earlier. In a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid Melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature.

Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations

Two sugarcane cultivars (R570 and SP71‐6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf‐free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane‐growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71‐6163 were infected by SCYLV genotypes BRA‐PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71‐6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.

Yellow leaf of sugarcane is caused by at least three different genotypes of sugarcane yellow leaf virus, one of which predominates on the Island of Réunion

The genetic diversity of sugarcane yellow leaf virus (SCYLV) was analyzed with 43 virus isolates from Réunion Island and 17 isolates from world-wide locations. We attempted to amplify by reverse-transcription polymerase chain reaction (RT-PCR), clone, and sequence four different fragments covering 72% of the genome of these virus isolates. The number of amplified isolates and useful sequence information varied according to each fragment, whereas an amplicon was obtained with diagnostic primers for 59 out of 60 isolates (98%). Phylogenetic analyses of the sequences determined here and additional sequences of 11 other SCYLV isolates available from GenBank showed that SCYLV isolates were distributed in different phylogenetic groups or belonged to single genotypes. The majority of isolates from Réunion Island were grouped in phylogenetic clusters that did not contain any isolates from other origins. The complete six ORFs (5612 bp) of five SCYLV isolates (two from Réunion Island, one from Brazil, one from China, and one from Peru) were amplified, cloned, and sequenced. The existence of at least three distinct genotypes of SCYLV was shown by phylogenetic analysis of the sequences of these isolates and additional published sequences of three SCYLV isolates (GenBank accessions). The biological significance of these genotypes and of the origin of the distinct lineage of SCYLV in Réunion Island remains to be determined.

P.306 Innate immune response in acute hepatitis E

The 5898 nucleotide single-strand RNA genome of Sugarcane yellow leaf virus (SCYLV) contains one long open reading frame, which is translated into a 120.6 kDa polyprotein. The sequences of SCYLV isolates from the two SCYLV-susceptible cultivars from Hawaii had a deletion of 48–51 nt in ORF1. SCYLV from 12 sugarcane hybrid cultivars from different origins were tested by RT-PCR using a specific set of primers, to investigate the genome segment for this deletion. Only three cultivars were found not to have the deletion (H87-4319, JA-605 and CP52-43), while SCYLV from nine cultivars (H73-6110, H87-4094, H78-7750, GT54-9, G84-47, H78-4153, H65-7052, C1051-73, Ph-8013) along with aphid (Melanaphis sacchari), which fed on SCYLV-infected H73-6110, contained a deletion of about 50 nt. The deleted sequence was located in the overlap frameshift of ORF1 and ORF2. Thus, ORFs 1 and 2 of SCYLV are translated via ribosomal frameshift and yield the 120.6 kDa viral replicase. ORF1 plays most likely a role in the replication and is a source of large variability among the virus population. To identify possible recombination events located in the RdRp domain of the Hawaiian isolates, two programs were used: RDP v.4.3 and RECCO. It is noteworthy that according both methods Haw73-6110 was found as a potential recombinant. On the other hand, opposed to the RDP package, RECCO revealed that Haw87-4094 isolate was also a recombinant whereas Haw87-4319 was not.