Immunosorbent Electron Microscopy Research Articles

Rotavirus-Associated Diarrhea in Outpatient Settings and Child Care Centers

To determine the prevalence of rotavirus infection in outpatient and child care center (CCC) settings during the seasonal rotavirus outbreak and to describe associated health care utilization. Prospective, multisite cohort study in various ambulatory settings. Participants were children with diarrhea (1) presenting to hospital emergency departments (EDs) and receiving intravenous (IV; n = 8) or oral (n = 1) hydration, (2) seen in pediatric practices (n=4), or (3) attending CCCs (n = 19) between November 1, 1997, and June 30, 1998. Prospective centralized testing of stool samples for rotavirus was performed using enzyme-linked immunosorbent assay and electron microscopy. Study nurses administered follow-up parent questionnaires for rotavirus-positive children. Prevalence of rotavirus-associated diarrhea. During the 8-month study, rotavirus was identified in 92 children with diarrhea: ED-IV, 20 (44%) of 45; ED-oral, 9 (47%) of 19; pediatric practices, 30 (20%) of 147; and CCCs, 33 (18%) of 186. Of 226 children with diarrhea in pediatric practices, all 5 who progressed to ED-IV hydration or hospitalization were tested, and 3 (60%) were rotavirus positive. Of 211 children in CCCs with diarrhea, 84% who required no health care visits were tested, and of these 10% were positive; of 56 who went on to require a health care visit and 8 who required ED-IV hydration or hospitalization, all were tested, and 27% and 75%, respectively, were rotavirus positive. Among 16 children with rotavirus followed up with ED-IV hydration, 4 (25%) returned and were hospitalized. Maximal health care intervention among 29 children with rotavirus enrolled in pediatric practices included 22 (76%) seeing the pediatrician only, 5 (17%) seeking further care in the ED, 1 (3%) receiving further ED-IV hydration, and 1 (3%) being hospitalized briefly. Maximal health care intervention for 33 children with rotavirus enrolled in CCCs included 13 (39%) who did not visit a physician, 11 (33%) who did, 3 (9%) who sought care in the ED, 1 (3%) who received ED-IV hydration, and 5 (15%) who were hospitalized. In CCCs, rates of diarrhea per 100 child-months of observation were as follows: ages 0 to 23 months, 6.6 episodes; ages 24 to 35 months, 1.9 episodes; and 3 years and older, 0.07 episodes; rates of rotavirus-associated diarrhea were as follows: ages 0 to 23 months, 1.1 episodes (28 of 2547); ages 24 to 35 months, 0.23 episodes (5 of 2185); and 3 years and older, 0 episodes (0 of 4124). Across a variety of outpatient and CCC settings, rotavirus is an important cause of diarrhea and a major cause of health care utilization.

First Report of Barley yellow streak mosaic virus-Infected Barley in Alaska.

Barley yellow streak mosaic virus (BaYSMV) was first described and reported in Montana and Alberta, Canada, more than 17 years ago (1). Since then, it has been detected in two other locations: Pocatello Valley, ID (3), and across the border in Utah. BaYSMV has now been found in the Alaskan interior. In July 1999, dry-land barley (Hordeum vulgare L.) growing in University of Alaska-Fairbanks experimental plots exhibited symptoms similar to those described for BaYSMV, including parallel chlorotic streaks and leaf banding. Mechanical inoculation of Nicotiana benthamiana with diseased barley sap produced systemic mosaic symptoms. As previously reported for BaYSMV sap-transmission tests (2), parallel inoculations to barley plants yielded no symptoms. Electron microscopy of leaf dips and minipurifications of infected N. benthamiana revealed long filamentous particles that matched the size and shape reported for BaYSMV (1). Ultrathin sections of diseased barley and N. benthamiana leaves displayed characteristic virus particles. BaYSMV was confirmed by immuno-sorbent electron microscopy assays (4) and western blot analysis with polyclonal antiserum. Long filamentous BaYSMV particles appeared only on grids coated with BaYSMV antiserum and exposed to diseased N. benthamiana sap. Total protein extracts from diseased barley tissue and inoculated N. benthamiana, as well as with protein extracted from partially purified preparations, were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigel and stained with Coomassie blue. Diseased samples, but not healthy controls, contained a protein of ≈33 kDa that was within the size range of a previously described protein from partially purified BaYSMV particles (2). Western blot analysis with an Immuno-Blot alkaline phosphatase assay system (Bio-Rad Laboratories, Hercules, CA) confirmed that the protein reacted with polyclonal BaYSMV. This is the first serological documentation of a BaYSMV-specific protein and that the ≈33-kDa protein is the main antigen recognized by the BaYSMV polyclonal antiserum. Based on virus particle shape and size, symptomology, mechanical transmission host range, and serology, we conclude that BaYSMV is associated with the barley disease observed. Barley yellow streak mosaic virus disease outbreaks are associated with recurring drought and are accompanied by infestations of the brown wheat mite vector, Petrobia latens Müller (1), so it is not surprising that this report coincides with abnormally dry conditions occurring throughout the 1990s in the interior of Alaska.

Mechanical transmission of maize rayado fino marafivirus (MRFV) to maize and barley by means of the vascular puncture technique

Maize rayado fino marafivirus (MRFV) was mechanically transmitted to kernels of three Costa Rican maize cultivars by the vascular puncture technique. A transmission rate of up to 43.2% was obtained for cultivar HC‐57. The rate of MRFV transmission to maize was possibly affected by the morphology of the kernel, which may influence physical access to the vascular tissue after water imbibition. Transmission to maize was confirmed by western blot and immunosorbent electron microscopy. By slight modifications of the procedure, MRFV was also transmitted to barley. This is the first report of MRFV infecting a species outside the supertribe Andropogonodae. Virus infection and replication in barley were confirmed by RT‐PCR detection of MRFV RNA and by detection of the virus by ELISA.

Transmission, Characterization, and Serology of a Luteovirus Associated with Yellow Leaf Syndrome of Sugarcane

ABSTRACT A previously uncharacterized luteovirus was associated with one form of yellow leaf syndrome (YLS), a widespread disease of sugarcane (Saccharum sp.). The virus was named Sugarcane yellow leaf luteovirus (ScYLV), and was identified in major sugarcane-producing areas of the world. Typical disease symptoms were reproduced when ScYLV was transmitted by Melanaphis sacchari or Rhopalosiphum maidis from infected to healthy sugarcane, suggesting that this virus may be the causal agent of one form of YLS. The only known hosts of ScYLV are Saccharum and Erianthus spp. Virions of ScYLV were 24 to 29 nm in diameter in sodium phosphotungstate at pH 5.0, had a buoyant density of 1.30 g/cm(3) in Cs(2)SO(4), and contained a 5.8-kb genomic ssRNA. The capsid protein had an estimated relative molecular mass of 27 kDa and was not glycosylated. A polyclonal rabbit antiserum raised against ScYLV did not detect any of eight other luteoviruses by enzyme-linked immunosorbent assay or immunosorbent electron microscopy, but in immunoblot assays, antibodies to ScYLV detected the RPV serotype of Barley yellow dwarf luteovirus. It is concluded that ScYLV is a previously undescribed luteovirus that is biologically and serologically distinct from other members of the group and may be the causal agent of one form of YLS of sugarcane.

Effective application of DAS-ELISA for detection of grapevine leafroll associated closterovirus-3 using a polyclonal antiserum developed from recombinant coat protein

A polyclonal antiserum (As163) specific to grapevine leafroll associated closterovirus-3 (GLRaV-3) was developed using a recombinant coat protein expressed in E. coli from a cDNA clone identified after immunoscreening of a cDNA library. Specificity of the antiserum to GLRaV-3 was shown by Western blot and immunosorbent electron microscopy. With this antiserum, an effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for GLRaV-3 detection. To evaluate the sensitivity of the antiserum in DAS-ELISA for virus detection, different combinations of antibodies were compared. Although best results were obtained when As163 was used for coating and a monoclonal antibody (MabNY1.1) was used as an enzyme conjugate, good results were also obtained when As163 was used both for coating and as an enzyme conjugate. Using this As163–Mab system in DAS-ELISA, we confirmed the presence of GLRaV-3 in a diverse collection of leafroll infected vines.

First Report of Soilborne Rye Mosaic Virus in Rye in Denmark.

Soilborne wheat mosaic furovirus (SBWMV)-like particles were detected in rye (Secale cereale) grown in sandy soil in West Zealand during spring 1999. Infected plants showed yellow leaf mosaic and light stunting. Electron microscopy of negatively stained crude sap preparations revealed rigid rod-shaped particles with two average lengths, 296 and 162 nm; average diameter was 23 nm. Sap-inoculation to Chenopodium quinoa and C. amaranticolor produced local leaf lesions when grown at 17°C but none when grown at 22 to 25°C. All the features agree with the description of SBWMV (1). Immunosorbent electronmicroscopy with polyclonal antiserum produced by W. Huth to furovirus-like particles isolated from rye in Germany gave a distinct decoration to particles. Light microscopy of roots cleared with 10% KOH and stained with a 0.5% solution of trypan blue in lactoglycerol revealed resting spores with a morphology and size similar to Polymyxa graminis, a furovirus vector. This is the first record of a furovirus on cereals in Denmark. The complete nucleotide sequence of the isolate was analyzed and compared with data on isolates from wheat. Sequence identity was only 74%. Therefore, the isolate was designated as soilborne rye mosaic virus. SBRMV has been recorded previously in rye and triticale in several regions of Germany (2).

Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy

Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6×10 15 particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3×10 15 particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.

First Report of Sugarcane Yellow Leaf Virus in the French West Indies.

Unusually severe leaf yellowing symptoms, similar to those described for yellow leaf syndrome (1), have been observed in several sugarcane clones in Guadeloupe since 1994, and since 1997 in Martinique. Leaf samples exhibiting various types of yellowing were taken from five different sugarcane clones, and analyzed by immunosorbent electron microscopy. Spherical particles, 24 to 28 nm in diameter and characteristic of luteoviruses, were found in two of five samples. The two infected samples showed yellowing on the underside of the midrib and one had a pinkish coloration on the upper side. The presence of sugarcane yellow leaf virus (ScYLV), the causal agent of sugarcane yellow leaf disease, was confirmed by reverse transcription-polymerase chain reaction (2) in these two samples and in 36 of 184 sugarcane clones bred in Guadeloupe and sent to Cirad's quarantine station in Montpellier, France. Following these observations, surveys were undertaken with a tissue blot enzyme immunoassay to analyze the distribution of ScYLV in sugarcane clones in the French West Indies. The midrib base of the first visible dewlap leaf was used to detect the presence of the virus in the phloem. In a first survey, clones of various origins worldwide were taken from germplasm collections. Two to three leaf samples per clone were analyzed from 78 clones in a collection in Guadeloupe and from 36 in a collection in Mar-tinique. Fifty of the 114 clones were infected by ScYLV, and ScYLV was detected in 21 of the 32 clones exhibiting severe leaf yellowing (score 3 or higher on a 1 to 5 scale). In a second survey, 19 leaf samples were taken from each of 53 clones from plants produced by Cirad's breeding program in Guadeloupe. The virus was detected in at least one sample for 25 of these 53 clones. ScYLV incidence in commercial fields was tested in Martinique in the variety B5992, which constitutes 57% of the cultivated area. Twenty leaves from different stools were sampled in six different fields, five of which had ScYLV-infected plants. The percentage of virus-infected stalks ranged from 0 to 90% whereas the percentage of stalks showing symptoms ranged from 50 to 100%. ScYLV appears widespread in the French West Indies, perhaps because a vector (Melanaphis sacchari) exists in Martinique and Guadeloupe. However, ScYLV was not found in all symptomatic plants, indicating that even if this luteovirus is a causal agent of leaf yellowing in the French West Indies, there may be other causal agents as well.

Two New Hosts for Poinsettia Mosaic Virus.

Poinsettia mosaic virus (PnMV), a possible member of the genus Tymovirus, commonly infects the potted flower crop Euphorbia pulcherrima Willd. ex Klotzsch (1). Two new host species for this virus were identified during grafting experiments with E. pulcherrima and other Euphorbia spp. E. cornastra (Dressler) A. Radcliffe-Smith was reciprocally grafted with PnMV-positive E. pulcherrima cv. Eckespoint Lilo. PnMV was detected by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) in E. cornastra leaves directly below the graft union 4 weeks after grafting. Infection was not fully systemic 6 weeks after grafting when screened by DAS-ELISA with antibodies specific to PnMV (DSMZ, Braunschweig, Germany). The symptomless infection in E. cornastra persisted in cuttings from grafted plants after a 1-year observation period. E. bubalina Boiss. anatomy differs from that of E. pulcherrima. The two species did not produce a viable graft union. However, in an experiment with two attempted graftings, the E. pulcherrima scions remained turgid for 14 to 18 days. As a result of one grafting, the E. bubalina rootstock tested positive for PnMV. The virus induced a mild mosaic in E. bubalina, but no reduction in growth. To confirm virus presence in E. cornastra and E. bubalina, both DAS-ELISA and immunosorbent electron microscopy were used. Non-grafted controls remained PnMV negative. PnMV was re-isolated from both species by sap inoculation to Nicotiana benthamiana. E. coulescens Haw., E. xylophyllides Brogn. ex Lem., E. marlothiana N. E. Br., and Ricinus communis L. were not infected by PnMV after similar grafting attempts. Reference: (1) A. A. Brunt et al., eds. 1996. Viruses of Plants. CAB Int., Wallingford, UK.

Evaluation of micropropagated plantain and banana (Musa spp.) for banana streak badnavirus incidence under field and screenhouse conditions in Nigeria

SummaryBetween 1991 to 1996, more than 50 Musa hybrids and 10 landraces were evaluated under field and screenhouse conditions for virus symptoms resembling those caused by banana streak badnavirus (BSV). The symptoms included chlorotic streaks, leaf deformation, stunting, cigar leaf death, distortion of the peduncle, bunch or fruits, and internal pseudostem necrosis. Immunosorbent electron microscopy (ISEM) of randomly selected plants with one or more of these symptoms confirmed the presence of BSV particles in 15 tropical Musa plantain hybrids (TMPx) and five Musa landraces. Under both field and screenhouse conditions, the incidence of symptomatic plants in the hybrids was significantly higher than in the landraces. The hybrids also generally had a higher concentration of BSV antigens, as determined by enzyme‐linked immunosorbent assay (ELISA). By contrast, most BSV‐infected landraces were symptomless and had very low or undetectable amounts of BSV antigens. There was a significant variation in incidence of symptomatic plants between genotypes, experiments and year of observation. These results are discussed in relation to the higher natural BSV incidence observed on some Musa hybrids as compared with their parental genotypes.

Occurrence of tomato spotted wilt tospovirus in horticultural crops and weeds in the Czech Republic in 1992/19971

Tomato spotted wilt tospovirus (TSWV) was found in 1992 in three glasshouses in the Czech Republic. Although the infected plants were destroyed, the virus was detected later in other glasshouses infested with Frankliniella occidentalis. The virus was identified on the basis of host range, enzyme‐linked immunosorbent assay (ELISA) and electron microscopy. The ornamental plant species most frequently infected with TSWV were Dendranthema morifolium and Zantedeschia sp. With regard to vegetable crops, TSWV infection was frequently detected in tomatoes and capsicums. The common weed hosts were Stellaria media and Galinsoga parviflora.

Truncated particles produced in fish surviving infectious hematopoietic necrosis virus infection: mediators of persistence?

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that produces an acute, lethal infection in rainbow trout (Oncorhynchus mykiss). Fish that survive infection cease to produce detectable infectious virus at approximately 46 days after infection, yet there is evidence that survivor fish continue to harbor virus particles (B. S. Drolet, P. P. Chiou, J. Heidel, and J. C. Leong, J. Virol. 69:2140-2147, 1995). In an effort to determine the biological function of these particles, the kidneys and livers from IHNV survivors were harvested and divided into samples for nested reverse transcriptase PCR analysis and explant culture. Sequences for the IHNV nucleoprotein and polymerase genes were detected in 50 and 89%, respectively, of the organs from survivor fish. When explant tissue cultures were infected with purified standard IHNV, the liver tissues from survivor fish produced up to 10-fold less virus than naive control fish liver tissues. In addition, immunosorbent electron microscopy analysis of the supernatant media from the cultured explants of survivor fish revealed truncated particles, whereas the control tissue supernatants contained only standard viral particles. These results suggest that the truncated IHNV particles observed in persistently infected fish are defective interfering particles that may mediate virus persistence.

Banana Streak Badnavirus and Cucumber Mosaic Cucumovirus in Farmers' Fields in Zanzibar.

Symptoms resembling those of viral leaf streak disease, caused by banana streak badnavirus (BSV), were observed in May 1998 on two banana (Musa spp.) landraces grown from farmer-collected propagules in a farmer's field at Kiboje Uchukuni, Zanzibar. Those showing symptoms were "French plantain" cv. Mzuzu and "Cavendish" banana cv. Mtwike. Leaf symptoms were expressed as chlorotic streaks and blotches. Leaf samples were indexed by immunosorbent electron microscopy with BSV and cucumber mosaic cucumovirus (CMV) antibodies, using partially purified preparations (2). The two landraces tested positive for BSV, corroborating the occurrence of BSV in Zanzibar. In addition, cv. Mtwike was found to be coinfected with CMV. No other viruslike particles were seen by electron microscopy. Although BSV has been reported in Zanzibar (1), it was only from symptoms in the Musa field genebank at Kizimbani Research Station. BSV has been found in many Musa collections worldwide, particularly in the widely grown cv. Mysore. This report confirms the presence of BSV in farmers' fields and is also the first report of CMV infecting banana in Zanzibar.

Transmission of Pineapple Mealybug Wilt-Associated Virus by Two Species of Mealybug (Dysmicoccus spp.)

ABSTRACT Closterovirus-like particles associated with mealybug wilt of pineapple were acquired and transmitted by the pink pineapple mealybug, Dysmicoccus brevipes, and the gray pineapple mealybug, D. neobrevipes. Mealybugs acquired pineapple mealybug wilt-associated virus (PMWaV) from infected pineapple plants or detached leaves. The virus was detected in plants by tissue blot immunoassay and confirmed by immunosorbent electron microscopy. Plants exposed to mealybugs reared on PMWaV-free pineapple tissue remained uninfected. The presence of ants was correlated with an increased rate of virus spread when caged with D. brevipes. All stages of D. neobrevipes acquired PMWaV, although vector efficiency decreased significantly in older adult females. The probability of a single third-instar immature transmitting the virus was 0.04. Both species of mealybug acquired and transmitted PMWaV from infected pineapple material that had been clonally propagated for decades, and both species acquired PMWaV from sources previously infected with the virus by the other mealybug species.

Improved purification and molecular properties of Omithogalum mosaic virus in Israel

Summary.Symptoms associated with infection by Omithogalum mosaic virus (OMV) were documented among horticulturally important Omithogalum spp. grown in commercial greenhouses and open fields in Israel. OMV was mechanically transmitted to Chenopodium quinoa and C. amaranticolor. The virus was purified from infected C. quinoa using CsCl step gradient centrifugation. Virus yield of 18–24 mg kg“1 were obtained. Antiserum produced to the purified virus was highly specific in immunoblots and immunosorbent electron microscopy. Reverse transcription ‐ polymerase chain reaction (RT‐PCR) utilising potyvirus‐specific primers and the viral RNA resulted in the amplification of a DNA product of 1275 nucleotides. This PCR product was cloned and sequenced; it encoded an open reading frame of 333 codons and a 3‘ non‐coding region of 275 nucleotides. Analysis of the amino acid sequences of the putative CP of the Israeli isolate of OMV showed 95% similarities to the South African isolate.

Characterization of a new potyvirus isolated from peanut (Arachis hypogaea)

During a survey of viruses of peanuts in South Africa a mechanically transmissible virus was isolated from a plant exhibiting chlorotic ringspots and blotches on the leaves. Typical potyvirus-like flexuous particles were detected by electron microscope examination. Pinwheel-shaped and laminated inclusions in ultrathin sections, reaction with a monoclonal antibody directed to a potyvirus common epitope, a single 33 kDa coat protein and aphid transmission using Myzus persicae all confirmed that the virus was a subdivision II member of the Potyviridae. Host range studies suggested that the virus was none of the previously reported potyviruses of peanuts or of subdivision II potyviruses. The serological relationships of the virus were studied using a range of 17 antisera to potyviruses in ELISA and immunosorbent electron microscopy (ISEM). The isolate reacted weakly with antisera to plum pox virus and bean yellow mosaic virus in ISEM only. Nucleotide sequence of a 624 bp DNA product was obtained following immuno-capture with a potyvirus common epitope antiserum, cDNA synthesis and PCR amplification with potyvirus specific primers which amplify the 3′ untranslated region and a part of the coat protein gene. The sequence was only distantly related to a number of potyviruses, whether amino acid or nucleotide sequences were used for comparisons. It is proposed that the virus be named peanut chlorotic blotch virus and be accepted as a new member of the genus Potyvirus in the family Potyviridae.

First Report of Celery Mosaic Potyvirus on Dill (Anethum graveolens).

In the spring and summer of 1997, nearly 90% of dill plants (Anethum graveolens L.) cultivated in a crop of Emilia-Romagna region (northern Italy), exhibited viruslike symptoms. Approximately 30% of the plants showed narrowing of the leaf lamina and stunting that evolved to yellow or bronze color. The plants appeared bushy and had low seed production. Electron microscopy of leaf-dip preparations revealed the association of flexuous, potyvirus-like particles of 750 nm in length with diseased dill plants. The identity of this virus was established by differential host reactions and serological assays. It was mechanically transmitted only to Chenopodium amaranticolor Coste et Reyn. and C. quinoa Willd. plants (which showed local chloro-necrotic lesions after about a week), and Petunia × hybrida Hort. Vilm.-Andr. plants (latently infected). No infections were obtained in several inoculated Umbelliferae plants, including dill and celery (Apium graveolens L.). Therefore, our isolate is similar to that described by others (1,2) and unlike the type strain that infects only members of the Umbelliferae. Virus particles reacted in protein A sandwich-enzyme-linked immunosorbent assay with antiserum to celery mosaic potyvirus (CeMV) (supplied from the Istituto di Fitovirologia Applicata, CNR, Turin, Italy). This result was further confirmed by immunosorbent electron microscopy decoration tests. CeMV in the field was likely transmitted to dill by abundant aphid populations from weeds and/or other Umbelliferae species cultivated in the same location. In particular, the weed host Daucus carota L. and the cultivated Petroselinum sativum Hoffm. plants both exhibited bronze foliage and stunting, and were found to be infected by CeMV. This note represents the first report of CeMV in A. graveolens.

First Report of Cassava Common Mosaic Potexvirus Infecting Chaya (Cnidoscolus chayamansa) in Tuvalu.

Chaya (Cnidoscolus chayamansa McVaugh) was introduced into Tu-valu as a leaf vegetable in the early 1990s (1) with the primary objective of alleviating vitamin A and C deficiencies, which at that time were prevalent throughout the population. Although there are no definitive quarantine records, we believe that the crop originated from vegetative cuttings introduced into Funafuti atoll from Kiribati. In the years following its introduction, chaya was distributed as cuttings taken from the original, introduced germplasm, to other islands and atolls of Tuvalu. The occurrence of yellow patches on the mature leaves of chaya cultivated in Funafuti had previously been attributed to iron deficiency, which was the inevitable consequence of growing the crop on coral atoll soils that are well known to be poor sources of metallic ions, notably iron, zinc, and copper. Following heavy cyclonic rains in March 1997, young, emerging chaya leaves were noted to exhibit an angular mosaic of the leaf lamina, characteristic of virus infections. Negatively stained sap from mosaic-affected leaves, when examined in the transmission electron microscope, was found to contain many rod-shaped virus particles similar in morphology to those of the Potexviridae (approximately 510 × 15 nm). The virus was transmitted mechanically to Chenopodium amaranticolor, in which it induced local lesion symptoms. The virus was found to be more closely related serologically to cassava common mosaic potexvirus (CsCMV) from Brazil than to that from Colombia, by immunosorbent electron microscopy with antisera kindly provided by F. Morales. Recently, CsCMV particles have been found in mosaic-affected chaya derived from the original Funafuti mother plants on Vaitupu Island. Similar symptoms have been observed on chaya being cultivated in Nui and Nanumaga islands. We conclude that CsCMV must now have been distributed throughout the atolls and islands of Tuvalu by infected planting material. We believe this to be the first report of the occurrence of CsCMV outside Central America and the first record of a plant virus from Tuvalu. Reference: (1) C. Howells and M. Bainbridge. 1996. South Pacific Commission, Pacific Island Forests and Trees. Page 10.

First Report of Hibiscus Chlorotic Ringspot Carmovirus in Tuvalu.

In September 1997, plants of Hibiscus manihot (locally called nambele) were observed on Vaitupu Island, Tuvalu, exhibiting an angular leaf mosaic and chlorosis that was not always clearly discernible. Electron microscopy of negatively stained sap from affected leaves revealed the presence of numerous isometric virus particles 28 nm in diameter. Poly-acrylamide gel electrophoresis of purified virus gave a single protein band of Mr 38,000 similar to that of the carmoviruses. Immunosorbent electron microscopy tests with antisera kindly provided by N. Spence showed the virus to be hibiscus chlorotic ringspot carmovirus (HCRSV) (1). This virus is also reported from El Salvador, the U.S., Australia, Thailand, Malaysia, Fiji, the Solomon Islands, and Vanuatu. It is not known how the virus reached Tuvalu but we suspect it was via infected cuttings, which were imported for the production of food supplements to combat acute deficiencies of vitamins A and C in the population. The virus is most likely to have been disseminated throughout the islands and atolls of Tuvalu through infected cuttings. Local spread within fields could occur through contaminated hands and cutting implements because of the ease with which the virus is mechanically transmitted. Reference: (1) H. E.Waterworth et al. Phytopathology 66:570, 1976.

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

SummaryA Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)‐ELISA, triple antibody sandwich (TAS)‐ELISA, antigen coated plate (ACP)‐ELISA, and protein‐A coated antibody sandwich (PAS)‐ELISA. TAS‐ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP‐ELISA and PAS‐ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV‐infected plants from asymptomatic plant samples than did TAS‐ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS‐ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV‐indexing of field samples, tissue sampling should be done during the rainy season when most BSV‐infected plants express severe symptoms.