|Maroon Lango, Clarissa|
|Guaragna, Mary Ann|
|Bandla, Murali - AGDIA,INC., INDIANA|
|Marquardt, Steve - AGDIA,INC., INDIANA|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 12, 2004
Publication Date: March 8, 2005
Citation: Maroon-Lango, C.J., Guaragna, M.A., Jordan, R.L., Hammond, J., Bandla, M., Marquardt, S.K. 2005. Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate. Archives of Virology. 150:1187-1201. Interpretive Summary: Pepino mosaic potexvirus (PepMV) was first found in pepino or pear melon (Solanum muricatum) in coastal Peru in 1974. PepMV reappeared early in 1999 in protected tomato (Lycopersicon esculentum) in the Netherlands and has since been reported to occur in tomato in several countries, including France, Germany, Spain, the Canary Islands, the United Kingdom, Italy, and Canada. The occurrence of Pepino mosaic virus in the US was first observed in tomato in Arizona in 2000 when a customer submitted samples of symptomatic tomato leaves apparently infected with a virus. Due to lack of a suitable serological test for PepMV at the time, samples, which tested negative by ELISA for other tomato viruses, were tested by PCR using generic Potexvirus group primer sets. The Potexvirus group PCR test did indicate the presence of a potexvirus in tomato, which was confirmed by subsequent cloning and sequencing of the PCR products. To date, PepMV has been detected in tomatoes growing in Arizona, California, Colorado, Florida, Maryland, New York, Ohio, Oklahoma, and Texas. Grower concerns prompted us to identify and characterize the virus in question. Only at hand, however, was the very small amount of leaf material that had been lyophilized for future ELISA testing. Here we describe the cloning and sequence analysis of two new strains of PepMV occurring in the US using the limited original lyophilized plant material source. This approach involved the use of very small amounts of total RNA obtained from infected leaves and fruits, and Potexvirus group and PepMV-specific PCR primers, and commercially available kits. Full-length sequence data on these two strains, PepMV-US1 and PepMV-US2, and sequence of the 3' end of a third strain isolated in Maryland, PepMV-US3, were obtained without propagation of the viruses in a suitable host and subsequent virus purification. We believe that this approach will be useful for viruses that occur in high titers in the host plant, and/or when a propagation host is not available for a given virus. Sequence analysis and pairwise comparisons between the US and European strains indicate that, while unique, PepMV-US1 is more closely related to the previously reported European strains than is PepMV-US2, whereas PepMV-US3 is nearly identical to the European isolates. The availability of coat protein- expressing clones of the two unique PepMV-US strains, also generated in this study, will allow us to develop highly discriminating monoclonal antibodies for more effective detection and as tools in epidemiological studies.
Technical Abstract: Three strains of Pepino mosaic virus (PepMV) found in the US have been cloned and sequenced. Full-length genomic sequences were obtained for two strains from Arizona, PepMV-US1 and PepMV-US2, from limiting amounts of the original co-infected tomato plant samples from Arizona. Sequence of the 3'-end of the third strain, PepMV-US3, came from infected tomato fruits from Maryland. Clones were generated by RT-PCR using total RNA from infected tissue as template and degenerate potexvirus and PepMV species and strain-specific primers. The genome organizations of PepMV-US1 and US2 were typical of potexviruses, with the following reading frame order: ORF 1 (US1: nt 86-4405, US2: nt 85-4401) encoding replicase with a predicted molecular weight of 163 kDa; ORF2 (US1: nt 4430-5134; US2: nt 4426-5130) encoding triple gene block protein (TGBp) 1 (26 kDa) ORF3 (US1: nt 5115-5486; US2: nt 5111-5482) encoding TGBp2 (14 kDa), ORF4 (US1: nt 5338'5586; US2: nt 5334'5582) encoding TGBp3 (9 kDa); and ORF5 (US1: nt 5633' 6346; US2: nt 5628-6341) encoding the coat protein (CP) (25 kDa). Gene-for-gene comparison between PepMV-US1 and US2 revealed the following amino acid identities: 91% in replicase, 89% in TGBp1, 92% in TGBp2, 85% in TGBp3, and 93% in the CP. Pairwise comparisons between the US and European strains indicate that, while unique, PepMV-US1 is more closely related to the previously reported European strains than is PepMV-US2. The CP of PepMV-US3 is nearly identical to the European isolates at the amino acid level. The serological reagents used in this study are able to detect all three US strains. The availability of clones of the expressible CPs of the two unique PepMV-US strains will allow us to develop highly discriminating monoclonal antibodies for more effective detection and as tools in epidemiological studies.