Role of extracellular vesicles during oocyte maturation and early embryo development.

The follicle is a dynamic microenvironment in the ovary where the oocyte develops. Intercellular communication between somatic cells and the oocyte inside the follicle is essential to generate a competent gamete. Extracellular vesicles are nanoparticles secreted by cells that mediate cell-to-cell communication in the follicle microenvironment and can be obtained from the follicular fluid. These extracellular vesicles have been studied as biomarkers and supplementation tools to mimic physiological conditions during assisted reproductive techniques because they are vehicles of bioactive molecules. Therefore, this paper reviews the importance of changes in the ovarian follicle and the effects of extracellular vesicles from follicular fluid during oocyte maturation and early embryo development. Finally, we propose that is important to consider the source of the extracellular vesicles to improve diagnostic methods and to increase invitro embryo production.

First Report of Tomato severe rugose virus in Chili Pepper in Brazil.

Three definitive and three tentative begomovirus species have been reported in tomato fields in Brazil according to a recent review (1). Extensive surveys have been conducted since the 1990s in solanaceous weeds and other crops planted close to tomato fields, but no tomato-infecting geminiviruses have been reported on those crops. During November 2003, leaves of one chili pepper plant "dedo-de-moça" (Capsicum baccatum var. pendulum) showing symptoms of yellow mosaic and leaf distortion were collected in Petrolina de Goiás (Goiás State). Serological analyses were carried out with polyclonal antisera produced in our laboratory against the following viruses: Potato virus Y (PVY), Pepper yellow mosaic virus (PepYMV), Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV), and Chrysanthemum stem necrosis virus (CSNV). Serological data showed that the plant was not infected with any of these viruses. A begomovirus-specific DNA-A fragment of 1.3 kb was amplified by polymerase chain reaction (PCR) from the analyzed plant. The fragment shared 98% identity to the partial coat protein coding region (CP), 94% to the intergenic region (IR), and 95% to the partial AC1 coding region of Tomato severe rugose virus (ToSRV) (GenBank Accession No. AY029750). Total DNA from the original infected plant was used to biolistically inoculate healthy plants of C. annuum and C. baccatum var. pendulum. Four resulting symptomatic plants, two from C. annuum and two from C. baccatum, were tested using PCR for begomovirus, and the nucleotide sequence of the amplified fragment confirmed they were infected with ToSRV. Whitefly inoculation of C. annuum, C. baccatum, and tomato was also performed, and all plants expressing symptoms were confirmed to be infected with ToSRV by sequencing a begomovirus-specific amplified fragment. Cloning of the complete DNA-A was achieved by using TempliPhi (Amersham Biosciences, Piscataway, NJ) amplification and digestion with a single cutting restriction endonuclease (2). Sequencing of several clones showed that the complete DNA-A (GenBank Accession No. DQ207749) was 97% identical to ToSRV, confirming the results of the previous PCR analysis. The deduced amino acid sequences showed identities of 97% to the CP, 95% to AC1, 96% to AC2, 96% to AC3, and 88% to AC4 of ToSRV. Although begomoviruses have not yet been causing any significant losses in chili pepper in Brazil, they may be of potential importance. Moreover, chili pepper, a plant commonly found in gardens throughout the country, may serve as an alternate host in tomato-producing areas. To our knowledge, this is the first report of a begomovirus infecting chili pepper in Brazil. References: (1) C. M. Fauquet et al. Arch. Virol. 148:405, 2003. (2). A. K. Inoue-Nagata et al. J Virol Methods 116:209, 2004.

A novel melon flexivirus transmitted by whitefly.

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.

Pepper yellow mosaic virus, a new potyvirus in sweetpepper, Capsicum annuum.

A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3' terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3'-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3'-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.

Sequence diversity of NS(M) movement protein of tospoviruses.

In order to determine the diversity of the movement protein (NS(M)) among tospoviruses, the NSM genes of five distinct tospovirus species occurring in Brazil (Tomato chlorotic spot virus, Groundnut ring spot virus, Chrysanthemum stem necrosis virus, Zucchini lethal chlorosis virus and Iris yellow spot virus) were cloned, sequenced and compared with NS(M) sequences of other available tospoviruses. The 'D-motif', a conserved region present in the majority of '30K superfamily' virus movement proteins, is present in all NSM amino acid sequences available. In addition to the 'D-motif', a conserved phospholipase A2 motif was found. The NSM amino acid sequence comparisons among tospovirus species revealed several conserved regions located in the internal part of the protein and diverse domains mainly located in the amino-terminus. Prediction of secondary structure showed similar patterns among all NS(M) proteins analyzed. Considering the geographical prevalence and phylogenetic analysis of N and NS(M) proteins, tospoviruses were tentatively clustered in 'American' and 'Eurasian' groups. Both phylogenetic trees may reflect the natural evolution of tospovirus species within distinct ecological niches. The sequence information obtained in this work would facilitate functional analysis of NS(M) during the tospovirus infection process.

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.

Coriander: A New Natural Host of Groundnut Ring Spot Virus in Brazil.

Coriander plants (Coriandrum sativum L. 'Palmeira'), showing stunting, chlorotic ring spots, necrosis, and malformation of apical leaves were observed on 50-day-old-plants in July 1998 in one seed production field at Petrolina, State of Pernambuco, Brazil, but not in nearby fields. Leaf samples were collected and tested by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with a panel of polyclonal antibodies made against the nucleocapsid protein (N) of tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), groundnut ring spot virus (GRSV), and impatiens necrotic spot virus (INSV) (1). All symptomatic samples reacted only with the GRSV antisera. Coriander leaf extracts from infected plants were mechanically inoculated onto potential indicator hosts. The virus induced systemic infection with vein clearing, chlorotic and necrotic spots, necrotic ring spots, mosaic, top distortion, and stunting within 21 days after inoculation on Capsicum annuum cv. Ikeda, C. chinense PI 159236, Physalis floridana, Nicandra physaloides, Nicotiana tabacum cv. TNN, N. benthamiana, Lycopersicon esculentum cv. Rutgers, Phaseolus vulgaris cv. BT2, and Gomphrena globosa. The symptomatic indicator plants tested positive for GRSV by DAS-ELISA. P. vulgaris, Chenopodium amaranthicolor, C. quinoa, and Cucurbita pepo (zucchini) cv. Caserta showed only small, necrotic, local lesions on inoculated leaves. Citrullus lanatus cv. Charleston Gray was asymptomatic. This is the first report of natural occurrence of GRSV on coriander in Brazil. Reference: (1) A. C. de Ávila et al. J. Gen. Virol. 71:2801, 1990.

Characterization of a Tospovirus Isolate of Iris Yellow Spot Virus Associated with a Disease in Onion Fields in Brazil.

A tospovirus from onion causing a disease known as "sapeca" by growers in Brazil was characterized. Symptoms on onion consisted of numerous eyelike spots on the leaves and flower stalks resulting in flower abortion. Nicotiana benthamiana and N. rustica were the only systemic hosts experimentally found. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) experiments demonstrated that this virus was serologically related to iris yellow spot virus (IYSV), a tospovirus recently described in the Netherlands. This virus, from onion, based on an amino acid sequence identity of 90.5% for the N gene protein, is regarded as a strain of IYSV and is designated IYSVBR This 10% divergence in the nucleocapsid protein may represent an adaptation of the virus to distinct ecological niches.

Widespread Occurrence of Tomato Geminiviruses in Brazil, Associated with the New Biotype of the Whitefly Vector.

Although tomato golden mosaic virus (TGMV) was reported in Brazil more than 20 years ago (3), tomato-infecting geminiviruses have not been of economic significance in the country until recently. However, a sharp increase in the incidence of geminivirus-like symptoms in tomatoes has been reported in several areas of Brazil since 1994. This has coincided with the appearance of the B biotype of Bemisia tabaci, which, as opposed to the A biotype, readily colonizes solanaceous plants (2). We have isolated geminiviruses from symptomatic tomato plants in the Federal District, in two different areas of the state of Minas Gerais, and in the state of Pernambuco. Tomato plants in these areas showed a variety of symptoms, including yellow mosaic, severe leaf distortion, down-cupping, and epinasty. Whitefly infestation was high in all fields sampled, and in some fields, particularly in Pernambuco, incidence of virus-like symptoms was close to 100%, and no tomatoes of commercial value were harvested (1). Using primer pairs PAL1v1978/PAR1c496 and PCRc1/PBL1v2040 (4), DNA-A and -B fragments were polymerase chain reaction (PCR)-amplified from total DNA extracted from diseased plants, cloned, and sequenced. Sequence comparisons of the PCR fragments indicated the existence of at least six different geminiviruses. The nucleotide sequence homologies for DNA-A fragments ranged from 67 to 80% for the 5' end of the cp gene, and from 44 to 80% for the 5' end of the rep gene. Data base comparisons indicated the viruses are most closely related to TGMV, bean golden mosaic virus from Brazil (BGMV-Br), and tomato yellow vein streak virus (ToYVSV), although homologies were less than 80% for the fragments compared. A similar lack of a close relationship with each other and other geminiviruses was obtained with two DNA-B component PCR products compared, corresponding to the 5' end of the BC1 open reading frame. Infectious, full-length genomic clones from the tomato viruses are being generated for biological and molecular characterization. References: (1) I. C. Bezerra et al. Fitopatol. Bras. 22:331, 1997. (2) F. H. França et al., Ann. Soc. Entomol. Bras. 25:369, 1996. (3) J. C. Matyis et al. Summa Phytopathol. 1:267, 1975. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Classification of tospoviruses based on phylogeny of nucleoprotein gene sequences.

The nucleotide sequences of the nucleoprotein (N) genes of seven tospovirus isolates representing three serogroups were determined and used to establish phylogenetic parameters to delineate species within the Tospovirus genus of the Bunyaviridae. A high sequence divergence (55.9% identity at the nucleotide level) was observed between isolates of serogroup I (tomato spotted wilt virus) and isolates of serogroup III (Impatiens necrotic spot virus). The serogroup II isolates take an intermediate position. Their N genes have 75% identity with those of serogroup I isolates and 57% with those of serogroup III isolates. Whereas the isolates within serogroups I or III have almost identical sequences, the two isolates BR-03 and SA-05 of serogroup II diverged significantly from each other (82.1% sequence identity). The results obtained support the conclusion that, in addition to the species TSWV and INSV, the serogroup II isolates BR-03 and SA-05 have to be considered as distinct species within the genus Tospovirus for which the names tomato chlorotic spot virus and groundnut ringspot virus, respectively, are proposed.

Distinct levels of relationships between tospovirus isolates.

The taxonomic relations of a number of tospovirus isolates, collected in different geographical areas and from different host plants, were studied. To delineate these isolates, properties such as susceptibility of a limited range of host plants, symptomatology, cytopathology, nucleocapsid composition, serology of their nucleocapsid proteins, and nucleotide sequence homology were compared. The results show that isolates which have previously been discriminated as members of three different serogroups, should in fact be regarded as representatives of at least three distinct virus species in the tospovirus genus.

Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions.

Defective interfering (DI) RNA molecules derived from the genomic L RNA segment of tomato spotted wilt virus (TSWV) were generated during sequential passage of the virus at high multiplicity. Characterization of DI RNAs from four distinct isolates by Northern blot analysis and sequence determination revealed that both the 5' and 3' genomic termini were retained in these molecules. Each DI RNA contained a single internal deletion of approximately 60% to 80% of the L RNA segment. All DI RNAs studied maintain an open reading frame (ORF) which suggests that these defective molecules should be translatable by ribosomes. Detection of only defective molecules with ORFs indicates either that association with ribosomes or translation is a prerequisite for the selection and maintenance of replicating DI RNAs, or that the truncated proteins produced play a role in their selection or replication. Analysis of the junction sites in the DI RNAs showed that short nucleotide sequences are repeated, one at the release and another at the reinitiation point on the L RNA. One of these is lost during the generation of the DI molecules. The presence of repeated sequences at the junction sites seems to be unique for tospovirus DI L RNAs; they have not been described for other DI systems of either positive- or negative-strand RNA viruses. A model for TSWV DI RNA generation is proposed in which the viral polymerase can 'jump' across the internal sequences from one secondary structure to another containing the repeated sequences, during the replication of the viral complementary L RNA segment.

Immuno-electron microscopical detection of tomato spotted wilt virus and its nucleocapsids in crude plant extracts.

Tomato spotted wilt virus (TSWV) particles were identified in thin sections and in crude extracts of leaves from plants infected with different TSWV isolates, using gold labelled protein A and antibodies prepared against purified virus particles or against nucleocapsid preparations. In addition, both in thin sections and in dip preparations aggregates were detected using either gold-labelled antiserum. These aggregates were not detected using antisera against envelope proteins or against the non-structural protein NSs. They were the only detectably labelled material in preparations made from plants infected by morphologically defective isolates of TSWV, i.e. isolates that did not produce enveloped particles. The aggregates were discerned in dip preparations as cloudy amorphous structures having a cottonwool boll-like appearance. These 'cotton bolls' were interpreted as being the in vitro structures of the nucleocapsid aggregates occurring in plant cells infected by isolates maintained by mechanical inoculation and are the only structures in cells infected by morphologically defective isolates (Ie, 1982; Kitajima et al., 1992). The detection of the 'cotton boll-like' structures in crude extracts provides an adequate and rapid method to establish the presence of nucleocapsid aggregates in cells of infected plants, irrespective of whether they are infected by morphologically defective isolates or not.

The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus.

Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.

Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage.

During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.

Serological differentiation of 20 isolates of tomato spotted wilt virus.

Twenty tomato spotted wilt virus (TSWV) isolates were serologically compared in ELISA employing five different procedures using a rabbit polyclonal antiserum against nucleocapsid proteins (NuAbR) and mouse monoclonal antibodies (MAbs), two directed to nucleocapsid proteins (N1 and N2) and four directed to glycoproteins G1 to G4. All the antisera were raised against TSWV-CNPH1. The 20 isolates were differentiated into two distinct serogroups. Serogroup I consisting of 16 isolates strongly reacted with NuAbR. The other four isolates were poorly recognized by NuAbR and were placed in another serogroup, designated II. The panel of MAbs differentiated the TSWV isolates into three serotypes. The 16 isolates forming serogroup I reacted strongly with the MAbs generated and were identified as serotype I isolates. The four isolates which made up serogroup II were split into serotypes II and III. The serotype II isolates did not respond or responded poorly with MAbs N1, N2 and G3. The two other isolates placed in serotype III were recognized by N1 but not by N2 and G3. Two isolates became defective after several mechanical passages and failed to respond or responded very poorly with MAbs directed to glycoproteins. Our results show that ELISA employing polyclonal and monoclonal antisera is a useful tool to differentiate TSWV isolates and to detect defective forms. The results also strongly suggest that TSWV nucleocapsid proteins are less conserved than the glycoproteins.