Tatsuya Nagata

Increase of Tospoviral Diversity in Brazil with the Identification of Two New Tospovirus Species, One from Chrysanthemum and One from Zucchini

ABSTRACT During a survey conducted in several different regions of Brazil, two unique tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of tospovirus species reported.

The N protein of Tomato spotted wilt virus (TSWV) is associated with the induction of programmed cell death (PCD) in Capsicum chinense plants, a hypersensitive host to TSWV infection.

In sweet pepper, the Tsw gene, originally described in Capsicum chinense, has been widely used as an efficient gene for inducing a hypersensitivity response (HR) derived Tomato spotted wilt virus (TSWV) resistance. Since previously reported studies suggested that the TSWV-S RNA mutation(s) are associated with the breakdown of Tsw mediated TSWV resistance in peppers, the TSWV genes N (structural nucleocapsid protein) and NS(S) (non-structural silencing suppressor protein) were cloned into a Potato virus X (PVX)-based expression vector, and inoculated into the TSWV-resistant C. chinense genotype, PI 159236, to identify the Tsw-HR viral elicitor. Typical HR-like chlorotic and necrotic lesions followed by leaf abscission were observed only in C. chinense plants inoculated with the PVX-N construct. Cytopathological analyses of these plants identified fragmented genomic DNA, indicative of programmed cell death (PCD), in mesophyll cell nuclei surrounding PVX-N-induced necrotic lesions. The other constructs induced only PVX-like symptoms without HR-like lesions and there were no microscopic signs of PCD. The mechanism of TSWV N-gene HR induction is apparently species specific as the N gene of a related tospovirus, Tomato chlorotic spot virus, was not a HR elicitor and did not cause PCD in infected cells.

A novel melon flexivirus transmitted by whitefly.

Summary.In recent years, a viral disease on melon plants has become a serious problem in Brazil. Symptoms were principally yellowing and mottling on older leaves. Long filamentous virus particles, resembling those of carlaviruses, were seen in symptomatic leaves. In this study, the 3′ terminal region of the virus genome isolated from an infected plant, including the last two ORFs, was cloned and sequenced. The sequence comprised a polyadenilated tail and two ORFs, one exhibiting similarity to potexvirus and carlavirus coat protein gene and the second to a carlavirus protein with potential nucleic acid-binding property. The sequence analysis, the genome organization and the particle morphology indicated that the virus could be classified as a novel whitefly-transmitted flexivirus. The name Melon yellowing-associated virus (MYaV) is tentatively suggested for this virus.

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

Multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species.

Primary cell cultures prepared from embryos of the thrips species Frankliniella occidentalis and Thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (TSWV). Using polyclonal antibodies against the viral nucleocapsid protein (N) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a F. occidentalis, and a T. tabaci population which failed to transmit the virus. The infection was found in approximately 40% of the monolayer cells. Using antibodies against a nonstructural protein (NSs) of TSWV, uniform and more diffused staining was observed throughout the cytoplasm of these cells, underlying active genome replication. The NSs protein accumulated slower than the N protein in the cells of both thrips species. No multiplication of TSWV was observed in a heterologous insect cell line, i.e. from Spodoptera frugiperda, suggesting the existence of specific host factors in the thrips-derived cells.

Bidens mosaic virus is a member of the Potato virus Y species.

The nucleotide sequence of the genomic 3′ terminal region (1,702 bases) of two Brazilian Bidens mosaic virus isolates (BiMV: BiMV-p and BiMV-b) was determined. BiMV-p and BiMV-b share 98% nucleotide sequence identity, and are most closely related to members of the potyvirus species: Sunflower chlorotic mottle virus (an isolate of Potato virus Y) and Potato virus Y. BiMV-p shares 88% capsid protein amino acid identity and 77% 3′UTR nucleotide sequence identity with SuCMoV an isolate of Sunflower chlorotic mottle virus. Phylogenetic analyses suggest the close evolutionary relationship of BiMV, SuCMoV and Potato virus Y (PVY), members of the PVY species. According to the analyses of capsid protein and 3′UTR sequences BiMV isolates must be regarded as a strain of Potato virus Y species, but their differences in host reactions and the phylogenetic distance suggest that they would be most likely better placed in a taxon between species and strains.

Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease.

BackgroundCitrus sudden death (CSD), a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the diseases distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV). CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms.ResultsA total of 286 RNA-dependent-RNA polymerase (RdRp) and 284 heat shock protein 70 homolog (HSP70h) gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h) was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD.ConclusionWe suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence) of a single CTV variant within these populations that triggers the disease.

Print-capture PCR for detection of tomato begomoviruses from plants and whiteflies

Print-capture (PC) Polymerase chain reaction (PCR) was evaluated as a novel detection method of plant viruses. Tomato (Lycopersicon esculentum) plants infected with begomovirus (fam. Geminiviridae, gen. Begomovirus) and viruliferous whiteflies were used to study the efficiency of the method. Print-capturing steps were carried out using non-charged nylon membrane or filter paper as the solid support for DNA printings. Amplified DNA fragments of expected size were consistently obtained by PCR from infected plants grown in a greenhouse, after direct application of printed materials to the PCR mix. However, virus detection from a single whitefly and from field-grown tomato samples required a high temperature treatment of printed material prior to PCR amplification. Comparison of nylon membrane and filter paper as the solid support revealed the higher efficiency of the nylon membrane. The application of print-capture PCR reduces the chances of false-positive amplification by reducing manipulation steps during preparation of the target DNA. This method maintains all the advantages of PCR diagnosis, such as the high sensitivity and no requirement of radioactive reagents.

Molecular diversity of ecologically distinct Mal de Río Cuarto virus isolates based on restriction fragment length polymorphism (RFLPs) and genome sequence analysis of segments 1, 7, 9 and 10

SummaryViruses of the species Mal de Río Cuarto virus (genus Fijivirus, family Reoviridae) cause significant economic losses in maize in Argentina. Genetic changes in the virus genome leading to better adaptation to diverse ecological conditions were postulated that would account for the increasing MRCV variability. The genomic differences between MRCV isolates from four ecologically different areas (Río Cuarto, RC; Pergamino, P; Jesús María, JM; and Tafí del Valle, TV) were studied. RT-PCR-amplified fragments comprising four genomic segments (Seg1, Seg7, Seg9 and Seg10) of MRCV isolates were compared by RFLPs and nucleotide sequences. The segments were chosen based on the proteins they encode: RNA-dependent-RNA polymerase, proteins putatively associated with tubular structures and viroplasm and the major outer capsid protein, respectively. Genetic comparison suggested that JM and TV isolates were genetically similar, but RC and P were different. Therefore, they were clustered in three genetic groups (JM = TV, RC and P). Together, nucleotide and amino acid sequence identities of the genomic segments were often above 96%. Seg1 was more variable (viral polymerase), whereas Seg7 (putative tubular structure) was the most conserved. Phylogeny analysis showed that MRCV isolates could be clustered in ‘mountain area’ and ‘high production area’ groups according to their geographical occurrence.

Diferenciação de estirpes de Potato virus Y (PVY) por RT-PCR

RT-PCR For differentiation of Potato virus Y strains in potato The Potato virus Y (PVY) has become the major virus problem in seed potato growing areas of Brazil. Only necrotic and ordinary PVY strains were found infecting potatoes in Brazil. This situation drastically changed around 1997 when an epidemic of a PVY necrotic variant causing necrotic rings on the potato tuber surface was observed in the country. This study aimed to test the tuber necrotic strain differentiation method proposed by Weilguny & Singh (1998). Twenty eight PVY isolates originated from infected tubers and leaves from four Brazilian States were analyzed by host reaction after inoculation in tobacco, by ELISA using polyclonal antiserum and by the 3-primer RT-PCR method. The ordinary strain type isolates induced vein clearing and chlorotic pearl spots on Nicotiana tabacum leaves. All 24 remaining isolates inducing vein necrosis in leaves were classified as necrotic strains. All 28 PVY isolates positively reacted to PVY polyclonal antiserum by Elisa. Three RNA extraction methods were compared and the guanidine hydrochloride method was the most efficient and of the lowest cost. One isolate from Santa Catarina State and three from Rio Grande do Sul out of 28 PVY isolates submitted to RT-PCR method were differentiated as PVY