First Report of Pepper mottle virus Infecting Tomato in Hawaii.

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5'- CACTCCCTATTATCCAGG(T)16-3') following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.

Control of papaya ringspot virus in papaya: a case study.

The papaya crop is severely affected by papaya ringspot virus (PSRV) worldwide. This review focuses on efforts to control the destructiveness of the disease caused by PSRV in Hawaii, starting from the use of cross protection to parasite-derived resistance with transgenic papaya expressing the PSRV coat protein gene. A chronology of the research effort is given and related to the development of technologies and the pressing need to control PSRV in Hawaii. The development of commercial virus-resistant transgenic papaya provides a tangible approach to control PSRV in Hawaii. Moreover, the development of transgenic papaya by other laboratories and employment of a mechanism of effective technology transfer to different countries hold promise for control of PSRV worldwide.

Field Resistance of Coat Protein Transgenic Papaya to Papaya ringspot virus in Jamaica.

Transgenic papayas (Carica papaya) containing translatable coat protein (CPT) or nontranslatable coat protein (CPNT) gene constructs were evaluated over two generations for field resistance to Papaya ringspot virus in a commercial papaya growing area in Jamaica. Reactions of R0 CPT transgenic lines included no symptoms and mild or severe leaf and fruit symptoms. All three reactions were observed in one line and among different lines. Trees of most CPNT lines exhibited severe symptoms of infection, and some also showed mild symptoms. R1 offspring showed reactions previously observed with parental R0 trees; however, reactions not previously observed or a lower incidence of the reaction were also obtained. The transgenic lines appear to possess virus disease resistance that can be manipulated in subsequent generations for the development of a product with acceptable commercial performance.

Update on the development of virus-resistant papaya: virus-resistant transgenic papaya for people in rural communities of Thailand.

Papaya (Carica papaya L.) is one of the most important and preferred crops in rural communities in Thailand. Papaya ringspot virus (PRSV) is a serious disease of papaya throughout Thailand. Efforts to control the virus by various methods either have not been successful or have not resulted in sustainable control. In 1995, collaborative research by the Department of Agriculture of Thailand and Cornell University to develop transgenic papaya resistant to PRSV was initiated. Two local Thai cultivars were transformed by microprojectile bombardment with the use of a nontranslatable coat protein gene of PRSV from Khon Kaen. Numerous kanamycin-resistantplants were regenerated and were inoculated with the PRSV Khon Kaen isolate for selection of resistant lines. Since 1997, promising RO transgenic lines have been transferred to the research station at Thapra for subsequent screenhouse tests and selection of the most PRSV-resistant lines. In selection set 1, three R3 lines initially derived from Khaknuan papaya showed excellent resistance to PRSV (97% to 100%) and had a yield of fruit 70 times higher than nontransgenic Khaknuan papaya. In selection set 2, one R3 line initially derived from Khakdam papaya showed 100% resistance. Safety assessments of these transgenic papayas have so far found no impact on the surrounding ecology. No natural crossing between transgenic and nonmodified papaya was observed beyond a distance of 10 m from the test plots. Analysis of the nutritional composition found no differences in nutrient levels in comparison with the nonmodified counterparts. Molecular characterization by Southern blotting revealed three copies of the transgene presented; however, no coat protein product was expressed. Data on additional topics, such as the effects offeeding the transgenic papaya to rats and the stability of the gene inserts, are currently being gathered.

Expression of the gene encoding the coat protein of cucumber mosaic virus (CMV) strain WL appears to provide protection to tobacco plants against infection by several different CMV strains.

The gene (cp) encoding the coat protein (CP) of cucumber mosaic virus (CMV) strain WL (CMV-WL, which belongs to CMV subgroup II) was custom polymerase chain reaction (CPCR)-engineered for expression as described by Slightom [Gene 100 (1991) 251-255]. CPCR amplification was used to add 5'- and 3'-flanking NcoI sites to the CMV-WL cp gene, and cp was cloned into the expression vector, pUC18cpexp. This CMV-WL cp expression cassette was transferred into the genome of tobacco (Nicotiana tabacum cv. Havana 423) via the Agrobacterium T-DNA transfer mechanism. R0 plants that express the CMV-WL cp gene were subcloned, propagated, and challenge-inoculated with CMV-WL. Several R0 plant lines showed excellent protection against CMV-WL infection; however, plants found to accumulate the highest CP levels did not show the highest degree of protection. Thus in our case, CP levels appear not to be a useful predictor of the degree of protection. Plants from the best protected CMV-WL cp gene-expressing R0 tobacco lines were also inoculated with CMV strains belonging to the other major CMV subgroup (subgroup I), CMV-C and CMV-Chi, and compared in a parallel experiment with a transgenic tobacco plant line that expresses the CMV-C cp gene. Plants expressing the CMV-WL cp gene appeared to show a broader spectrum of protection against infection by the various CMV strains than plants expressing the CMV-C cp gene.

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression.

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination.

Leafroll Virus Is Common in Cultivated American Grapevines in Western New York.

American grapevines (Vitis labrusca L. 'Niagara'; Vitis × labruscana L. H. Bailey 'Concord' and 'Catawba'; V. labrusca × V. riparia Michx. 'Elvira') from 24 vineyards in the New York portion of the Lake Erie production region (>13,000 ha cultivated) were tested to explore a possible relationship between virus infection and an unexplained fruit set malady in the district. One-year-old cane segments were collected 4 to 6 weeks before budbreak from 65 individual vines, which previously had been identified as malady positive or negative. Preparations from bark scrapings were tested for the presence of double-stranded (ds) RNA and for fan leaf degeneration virus, tobacco streak virus, and grapevine leafroll associated closterovirus-3 (GLRaV-3) by enzyme-linked immunosorbent assay (ELISA). Mechanical transmission of other potential viruses to Chenopodium quinoa was attempted with sap extracted from young shoots forced from intact segments of sampled canes. GLRaV-3 was detected in 17 (26%) of the sampled vines from eight (33%) of the vineyards, but there was no apparent relationship between infected vines and the fruit set malady. Vines of all four cultivars were infected. dsRNA was detected in all 17 samples positive for GLRaV-3 plus four additional samples. No other viruses were detected. Near harvest, nine vines (from two vineyards) previously testing positive for GLRaV-3 were examined and retested; all nine tested positive again, although none showed any overt symptoms of viral infection. This is believed to be the first report of GLRaV-3 from American grape vineyards in New York. The source of these infections is unknown: all vines were self rooted, the individual vineyards had been planted independently at different times, and V. vinifera and its hybrids are rare in the district. Wild grapevines (primarily V. riparia) are abundant in the region, although it has been reported that leafroll disease does not occur naturally in wild North American grapes (1). Nevertheless, our results indicate that cultivated American grapevines can be common reservoirs of GLRaV-3, and furthermore suggest the need to reassess the possibility that wild grapes also may serve as reservoirs of the virus. Trials are currently underway to determine possible effects of GLRaV-3 on cv. Concord, the most widely planted variety in the region. Reference: (1) A. C. Goheen. 1988. Leafroll. Page 52 in: Compendium of Grape Diseases. R. C. Pearson and A. C. Goheen, eds. American Phytopathological Society, St. Paul, MN.

Virus Coat Protein Transgenic Papaya Provides Practical Control of Papaya ringspot virus in Hawaii.

Since 1992, Papaya ringspot virus (PRSV) destroyed nearly all of the papaya hectarage in the Puna district of Hawaii, where 95% of Hawaii's papayas are grown. Two field trials to evaluate transgenic resistance (TR) were established in Puna in October 1995. One trial included the following: SunUp, a newly named homozygous transformant of Sunset; Rainbow, a hybrid of SunUp, the nontransgenic Kapoho cultivar widely grown in Puna, and 63-1, another segregating transgenic line of Sunset. The second trial was a 0.4-ha block of Rainbow, simulating a near-commercial planting. Both trials were installed within a matrix of Sunrise, a PRSV-susceptible sibling line of Sunset. The matrix served to contain and trace pollen flow from TR plants, and as a secondary inoculum source. Virus infection was first observed 3.5 months after planting. At a year, 100% of the non-TR control and 91% of the matrix plants were infected, while PRSV infection was not observed on any of the TR plants. Fruit production data of SunUp and Rainbow show that yields were at least three times higher than the industry average, while maintaining percent soluble solids above the minimum of 11% required for commercial fruit. These data suggest that transgenic SunUp and Rainbow, homozygous and hemizygous for the coat protein transgene, respectively, offer a good solution to the PRSV problem in Hawaii.

A three-year report of the medical helicopter transportation system of Connecticut.

Over 63% of the patients transported by the LIFE STAR crew are the victims of trauma. The system has transported 2,215 ill and/or injured patients, the majority of whom are critical, either from the scene of an injury or from a medical facility to another of greater specialization. With the audit procedure indicating a 95% positive predictive value for summoning this service, LIFE STAR has contributed to the care of critically ill or injured persons in Connecticut and surrounding areas.

Different mechanisms protect transgenic tobacco against tomato spotted wilt and impatiens necrotic spot Tospoviruses.

We generated transgenic tobacco plants expressing the sense or antisense untranslatable N coding sequence of the lettuce isolate of tomato spotted wilt virus (TSWV-BL) as well as transgenic plants containing the promoterless N gene of the virus. Both sense and antisense untranslatable N gene RNAs provided protection against homologous and closely related isolates but not against distantly related Tospoviruses. These RNA-mediated protections were most effective in plants that synthesized low levels of the respective RNA species and appears to be achieved through the inhibition of viral replication. Unlike the sense RNA-mediated protection, the level of the antisense RNA-mediated protection depended on the concentration of the inoculum and the size of the test plants. Comparisons with previous results in transgenic plants expressing the intact N gene suggest that resistance to homologous and closely related TSWV isolates in plants that express low levels of the translatable N gene is due to the presence of the N gene transcript and not the N protein. In contrast, resistance to distantly related Tospoviruses is due to accumulation of high levels of the N protein and not due to the presence of the N gene transcript.

Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene.

We obtained transgenic tobacco plants expressing the papaya ringspot virus (PRV) coat protein (CP) gene by transformation via Agrobacterium tumefaciens. Expression was effectively monitored by enzyme-linked immunosorbent assays (ELISA) of crude tissue extracts. Subcloned plants derived from eight original Ro transformants were inoculated with potyviruses: tobacco etch (TEV), potato virus Y (PVY), and pepper mottle (PeMV). Plants that accumulated detectable levels of the PRV CP showed significant delay in symptom development and the symptoms were attenuated. Similar results were obtained with inoculated R1 plants. We conclude that the expression of the PRV CP-gene imparts protection against infection by a broad spectrum of potyviruses.

Varying genetic diversity of Papaya ringspot virus isolates from two time-separated outbreaks in Jamaica and Venezuela.

Coat protein sequences of 22 Papaya ringspot virus isolates collected from different locations in Jamaica and Venezuela in 1999 and 2004, respectively, were determined and compared with sequences of isolates from earlier epidemics in 1990 and 1993. Jamaican isolates collected in 1999 exhibited nucleotide sequence identities between 98 and 100% but shared lower identities of 92.2% with an isolate collected in 1990. Isolates from the 2004 epidemic in Venezuela exhibited more heterogeneity, with identities between 88.7 and 98.8%. However, isolates collected in 1993 were more closely related (97.7%). The viral populations of the two countries are genetically different and appear to be changing at different rates; presumably driven by introductions, movement of plant materials, geographical isolation, and disease management practices.

Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein.

The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.

Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants.

RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic Nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387-453 bp) of the N gene displayed resistance through post-transcriptional gene silencing. Although smaller N gene segments (92-235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (i) a critical length of N transgene (236-387 bp) is required for a high level of transgene expression and consequent gene silencing, and (ii) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.

Nucleotide sequences of the coat protein genes and flanking regions of cucumber mosaic virus strains C and WL RNA 3.

Several strains of cucumber mosaic virus (CMV) have been classified, and nucleic acid hybridization data indicate that these strains differ widely in nucleotide sequence. We have constructed cDNA clones of the coat protein coding regions of CMV strains C and WL, and have compared the nucleotide sequences of the RNA 3 intergenic region, coat protein gene, and 3' untranslated region with published CMV sequences from the same regions of the Q, D and Y strains. These comparisons show that the C and WL strains belong to different CMV subgroups, and that the subgroups are more closely related in sequence than suggested by previous nucleic acid hybridization studies.

Rupestris stem pitting associated virus-1 consists of a family of sequence variants.

Rupestris stem pitting (RSP) seems to be one of the most widespread virus diseases of grapevines. A virus, designated as rupestris stem pitting associated virus-1 (RSPaV-1), is consistently associated with, and likely to be the causative agent of RSP. Sequence analyses of cDNA clones derived from several RSP-affected grapevines suggested that a family of sequence variants of RSPaV-1 was associated with RSP. The genome structure of the sequence variants is identical to that of RSPaV-1 in that they had five open reading frames (ORF) and sequence identities ranging from 75 to 93% in nucleotide sequence and from 80 to 99% in amino acid sequence. ORF5 (coat protein) and the carboxyl-terminal portion of ORF1 (replicase) appeared to be the most conserved regions. The coat proteins of the sequence variants exhibited highly similar antigenic indices, suggesting serological relatedness among them. The cDNA clones obtained through reverse transcription-polymerase chain reaction from RSP-infected grapevines were heterogeneous in nt sequence with identities of 77-99% relative to RSPaV-1. Furthermore, a number of sequence variants were identified in several grapevines infected with RSP. Baselines for defining RSPaV-1 and possible mechanisms accounting for infection of grapevines with multiple sequence variants of RSPaV-1 are proposed. Findings from this study should have practical applications toward understanding the etiology of RSP and developing reliable assays to rapidly detect the disease.

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos.

Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 µg·ml(-1) kanamycin. Four putative transgenic plant lines were obtained from the cp gene(-) vector and two from the cp gene(+) vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene(+) plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.

Watermelon mosaic virus II and zucchini yellow mosaic virus: cloning of 3'-terminal regions, nucleotide sequences, and phylogenetic comparisons.

The 3'-terminal genomic regions of an isolate of watermelon mosaic virus II (WMVII) and a Florida isolate of zucchini yellow mosaic virus (ZYMV-F) have been cloned. The nucleotide sequence of the WMVII cDNA clone shows the presence of the large nuclear inclusion protein gene, the coat protein gene and 3' untranslated region. The nucleotide sequence of a ZYMV-F cDNA clone shows the presence of the coat protein gene and 3' untranslated region. Comparisons of the nucleotide and deduced amino acid sequences of these clones with those from other potyviruses show that WMVII and the soybean mosaic virus N strain are closely related, thus supporting their classification as different strains of the same virus. Our comparisons also indicate that ZYMV-F is a distinct potyvirus type and that its closest relative is WMVII. Phylogenetic analysis using the most-parsimonious branching arrangement derived from the alignment of coat protein gene sequences suggests the existence of two major potyvirus groupings.

Determination of whether tomato spotted wilt virus replicates in Toxorhynchites amboinensis mosquitoes and the relatedness of this virus to phleboviruses (family Bunyaviridae).

Tomato spotted wilt virus (TSWV) has been reported to be morphologically, molecularly and structurally similar to viruses in the family Bunyaviridae. By various types of enzyme-linked immunosorbent assays (ELISA) and Western blot hybridizations, we tested TSWV with antibodies to 12 viruses in the Phlebovirus genus of this family. Serological relatedness was not found between TSWV and phleboviruses. However, one preparation of antibody to Arumowot virus reacted with a 53-kD protein from healthy plant extracts. Six-day-old adult Toxorhynchites amboinensis mosquitoes were inoculated with purified TSWV. Infectious virus was not detected in any of the injected insects during the 5-week test period. However, TSWV antigens were detected in these mosquitoes by ELISA at the original injected level for at least a week after injection. TSWV antigen concentration began to decrease thereafter, but remained at detectable levels for as long as 5 weeks after injection. However, there was no evidence that TSWV replicated in mosquitoes.