Papaya Ringspot Virus Isolates Research Articles

Development of Recombinant Coat Protein Antibody Based IC-RT-PCR and Comparison of its Sensitivity with Other Immunoassays for the Detection of Papaya Ringspot Virus Isolates from India

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

GENETIC DETERMINANT OF PAPAYA RINGSPOT VIRUS FOR INFECTION OF PAPAYA

Papaya ringspot virus (PRSV) is a major limiting factor for large-scale planting of papaya and cucurbits. Based on the difference of host range, most strains of PRSV belong to type P (PRSV P, papaya infecting) devastating on papaya in tropical areas or type W (PRSV W, non-papaya-infecting) causing severe loss on cucurbits worldwide. Viruses of PRSV P infect plants of the families Chenopodiaceae, Cucuribitaceae and Caricaceae (papaya family), whereas, viruses of PRSV W only infect plants of Chenopodiaceae and Cucuribitaceae. Phylogenetic analyses of PRSV isolates revealed that the divergence of PRSV is correlated to geographic distribution and not to the biological difference in host reactions, suggesting that P type viruses may derive from W type viruses. To study the genetic determinant(s) of PRSV for papaya infection, infectious cDNA clones of type P and type W viruses, driven by a bacteriophage promoter to express in vitro infectious transcript or by Cauliflower mosaic virus 35S promoter to express in vivo infectious transcript, were generated in our laboratory and used for recombination analyses. The bioassay of recombinant viruses revealed that the nt 6500-7000 of PRSV P genome is responsible for papaya infection. Sequence comparison showed that amino acid positions at 176 of NIaVPg, and 27 and 205 of NIaPro are significantly different among type P and type W viruses. Site-directed mutagenesis indicated that Lys 27 of NIaPro determines host specificity of PRSV for infection of papaya. Our results indicate that the host range of PRSV can be switched just by a single amino acid mutation. Thus, we have shown experimental evidence that PRSV type P may easily evolve from type W and explained the reason why type P and W viruses are closer to each other in the same geographic zone. Furthermore, the potential risk for planting papaya adjacent to cucurbit fields is also discussed.

Comparative genomics of Papaya ringspot virus pathotypes P and W from India

Papaya ringspot virus (PRSV), a definite member of the family Potyviridae and the genus Potyvirus [5], occurs worldwide and is a major constraint to papaya (Carica papaya) and cucurbits production throughout the tropical and subtropical regions [21]. The virus is readily vectored by aphids in a non-persistent manner [16]. The virus was first recorded from western India in 1958 [4] and since then it has spread to different geographical locations, limiting papaya and cucurbits production [1, 8, 9]. Prevalence of both papaya-infecting (Type P) and non-papaya-infecting (Type W) pathotypes has been recorded. The virus induces variety of symptoms on foliage, stem and fruit in papaya and cucurbits. PRSV has flexuous filamentous particles (760–800 nm 9 12 nm) with a single-stranded, positivesense RNA genome of approximately 10,000 nucleotides. Fifteen PRSV isolates belonging to different geographical locations have been completely sequenced. PRSV isolates from India have been characterized for coat protein only [1, 8, 9], and information on their complete genome sequences is lacking. In this study, complete genome sequences of pathotypes P and W from India and their sequence divergence, phylogenetic relationships and recombination with 15 other PRSV genomes have been determined to investigate their origin and host specificity. Preliminary results on pathotype P have been published [15]. PRSV isolates from symptomatic papaya (Carica papaya) (pathotype P) and spongegourd (Luffa aegyptiaca) (pathotype W) plants were collected from the Indian Agricultural Research Institute experimental farm in New Delhi and propagated in papaya (cv. Pusa Nanha) and pumpkin (Cucurbita pepo), respectively, in a glass house through sap inoculations using 0.1 M phosphate buffer, pH 7.2, containing 0.1% 2-mercaptoethanol and celite as an abrasive. The details of the PRSV genome sequences collected from GenBank (http://www.ncbi.nlm.nih.gov) are shown in Table 1. The analysis utilized the nucleotide and translated amino acid sequences of the whole genome or of individual cistrons. The complete genome sequence of PRSV RNA was generated with nine overlapping fragments using nine pairs of specific primers. Viral RNA was reverse transcribed and amplified using high-fidelity Taq DNA polymerase (New England Biolabs). Two clones of each of the nine amplified products (700–1,900 bp) were sequenced in both (30 and 50) directions with 100% identity in the overlapping regions. The sequences of overlapping cloned fragments that were generated were assembled manually using the BioEdit software version 5.09.04 [7]. Nucleotide and amino acid sequences representing the complete genomes of pathotypes P and W and their cistrons were aligned using CLUSTAL W [19]. A sequence similarity matrix between polyproteins and specific cistrons was calculated using the BioEdit software version 5.09.04 [7]. Recombination sites were identified using RDP [11], GENECONV [14], BOOTSCAN [12], MAXIMUM CHISQUARE [13], CHIMAERA [12] and SISTER SCAN [6], the non-parametric recombination detection methods implemented in RDP2 [12]. A multiple comparison corrected P-value cutoff 0.05 and default settings were used throughout, and only events detectable by two or more different methods were retained for further analysis. S. K. Mangrauthia Plant Pathology Section, Directorate of Rice Research, Hyderabad, India

Complete genomic sequence of a Papaya ringspot virus isolate from Hainan Island, China

Papaya ringspot virus (PRSV, genus Potyvirus, family Potyviridae) causes significant yield losses in papaya and in some cucurbits throughout tropical and subtropical regions [7]. Based on its host specificity, PRSV is classified into two biotypes. Type W is known to infect only cucurbits, whereas type P infects both papaya and cucurbits. The first PRSV genome completely sequenced and characterized was the Hawaiian isolate, PRSV-H [11]. The complete sequences of a number of PRSV isolates have since been determined [2, 3, 5, 6, 10]. Hainan Island, having 1,300 ha of papaya plantation and an annual output of 70,000 tons, is the most important papaya-producing region in China. The papaya ringspot disease has been and will remain a major challenge in papaya production in Hainan. The disease in Hainan displayed typical symptoms of PRSV infection [11]. However, the causative virus and its relationship with other PRSV isolates have not been characterized. Understanding the exact nature of the PRSV in Hainan is important for the management of the disease. This importance is apparent in light of recent reports that described different specificities of PRSV resistance displayed by transgenic papaya lines expressing different PRSV CP genes toward PRSV isolates from different geographic origins [8, 9]. In this paper, we report the complete genomic sequence of PRSV (PRSVHN) from the Hainan Island, China, and present evidence that PRSV-HN represents a unique and novel PRSV isolate.

Papaya ringspot virus‐P: characteristics, pathogenicity, sequence variability and control

Papaya ringspot virus (PRSV) is an aphid-transmitted plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV isolates belong to either one of two major strains, P or W. The P strains infect both papaya and cucurbits whereas the W strains infect only cucurbits. PRSV-P is found in all major papaya-growing areas. Virions are filamentous, non-enveloped and flexuous measuring 760-800 x 12 nm. Virus particles contain 94.5% protein and 5.5% nucleic acid. The protein component consists of the virus coat protein (CP), which has a molecular weight of about 36 kDa as estimated by Western blot analysis. Density of the sedimenting component in purified PRSV preparations is 1.32 g/cm(3) in CsCl. The PRSV genome consists of a unipartite linear single-stranded positive sense RNA of 10 326 nucleotides with a 5' terminus, genome-linked protein, VPg. The virus is naturally transmitted via aphids in a non-persistent manner. Both the CP and helper component (HC-Pro) are required for vector transmission. This virus can also be transmitted mechanically, and is typically not seed-transmitted. PRSV has a limited number of hosts belonging to the families Caricaceae, Chenopodiaceae and Cucurbitaceae. Propagation hosts are: Carica papaya, Cucurbita pepo and Cucumis metuliferus cv. accession 2459. Local lesion assay hosts are: Chenopodium quinoa and Chenopodium amaranticolor. Two transgenic papaya varieties, Rainbow and SunUp, with engineered resistance to PRSV have been commercially grown in Hawaii since 1998. Besides transgenic resistance, tolerant varieties, cross-protection and other cultural practices such as isolation and rogueing of infected plants are used to manage the disease. 00.057.0.01.045. 57010045. http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010045.htm.

Erratum to: Molecular characterization of a severe isolate of papaya ringspot virus in Mexico and its relationship with other isolates

In the online and print version of the original article, the corresponding author’s previous email address is no longer available. The updated email address is: lsilva@ira.cinvestav.mx

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.

Complete genome sequence of an isolate of Papaya ringspot virus from India

Complete genome sequence of an isolate of Papaya ringspot virus from India

Molecular characterization of a severe isolate of papaya ringspot virus in Mexico and its relationship with other isolates

The virus most often reported in papaya (Carica papaya L.) is papaya ringspot (PRSV). The aim of this work was the molecular genomic characterization of a Mexican severe isolate of PRSV-P "Mex-VrPO" (isolate from the State of Veracruz in Paso de Ovejas) as well as its comparison with other isolates from other world regions. The linear, assembled, single-strand positive sense RNA genome of PRSV-P Mex-VrPO was 10320 nt in length (excluding the poly(A) tail) and contained a single large predicted ORF with 3344 aa. The comparative analysis of our PRSV isolates and five others reported before, showed the most variable proteins were P1, P3, 6 K and CP with 13-33%, 5-7%, 6-9% and 5-9% divergence respectively. The most conserved ones were CI, NIb and HC-Pro (2-3%, 3-5% and 4-5%). The phylogenetic analysis showed a close relation between the Mexican (Mex-VrPO) and Hawaiian (PRSV-P HA) isolates. This work provided the first opportunity to establish the foundation for (1) understanding whole genome and polyprotein variability between Asian and American PRSV isolates, and (2) elucidating major trends in the relative evolution of viral cistrons as deduced from in silico recombination analyses.

Distribution of Papaya ringspot virus and Papaya mosaic virus in Papaya Plants (Carica papaya) in Mexico.

We report the results of a survey for the presence of Papaya ringspot virus (PRSV) along the coasts of the Gulf of Mexico and the Pacific Ocean, in 15 federal states of Mexico that account for over 98% of the national papaya production. More than 80 locations were visited in 58 counties. Out of a total of 267 papaya leaf samples, 157 tested positive for PRSV. We tested for the presence of three other viruses because of the occurrence of severe, atypical symptoms in plantations. Only Papaya mosaic virus (PapMV) was detected. PRSV was present in every county. PapMV was less frequent, but its overall distribution was almost identical. PRSV and PapMV occurred in single or mixed infections of papaya and other host species that could function as virus reservoirs. We investigated the diversity of the coat protein (CP) sequences of 36 PRSV isolates. The amino acid sequence divergence among all isolates ranged from 0.4 to 9.9%, and was comparable to that found in other regions of the world. In contrast to most of these world regions, there is a clear correlation between CP sequence variation and the geographical origins of the virus isolates.

(283) Biolistic Genetic Transformation of Carica papaya L. using the Helios™ Gene Gun

Biolistic genetic transformation of plants with viral genes is a method for controlling plant virus diseases; however, optimization of the particle bombardment parameters according to the transformation system is a key factor for an appropiate transgene expression and, therefore, a stronger resistance mechanism in transgenic plants. In order to optimize biolistic parameters, somatic papaya (Carica papaya L.) cv. Maradol embryo masses were bombarded with the CAMBIA 1301 plasmid construction that contains the coat protein gene (CP) of the papaya ringspot virus isolate of Colima, Mexico, driven by the double constitutively CaMV 35S promoter and flanked for the GUS and hygromycin (hpt) resistance genes. Particle bombardment protocol was carried out using the Helios™ Gene Gun device (BioRad) and the manufacturer's instruction manual. Helium pressure (50, 100, and 150 psi) and gold particle size (0.6, 1.0, and 1.6 μm) were evaluated. Five days after bombardment, somatic embryo clusters were used for GUS transient expression and, during 2 months, were selected into 50, 75, and 150 mg·L-1 hygromycin-containing media to its later CP-PCR detection. Results showed that 50 psi and 1.0 μm were the two optimal values for the assayed analyses. This is the first report of genetic transformation of papaya using the Helios™ Gene Gun device as a new tool compared to conventional PDS-1000/He.

Influence of Coat Protein Transgene Copy Number on Resistance in Transgenic Line 63-1 against Papaya Ringspot Virus Isolates

Line 63-1 is a `Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R1 plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thailand (TH), and Brazil (BR). Genetic and molecular data obtained in this study confirm that line 63-1 has two CP transgene insertion sites; segregation analysis shows that the CP and the npt II genes are present at both loci. To study the potential effect of gene dosage on resistance, various populations of R1, R2, and R3 seedlings were challenged by PRSV HA, BR, and TH. A R1 population obtained by self-pollination of line 63-1 hermaphrodite R0 plant exhibited resistance to all three isolates. The percentage of plants resistant to all three PRSV isolates increased in 63-1-derived populations as a result of recurrent selection. Additional genetic studies demonstrate that the number of resistant plants in a 63-1-derived population is directly correlated with the number of plants with multiple transgene copies. We conclude that transgene dosage plays a major role in affecting the resistance of 63-1 to PRSV isolates from various geographical locations.

Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus

Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform secondary somatic embryo cultures derived from immature zygotic embryos. Fifty-four transgenic lines, 26 translatable and 28 nontranslatable gene versions, were regenerated, with a transformation efficiency of 2.7%. Inoculation of cloned R0 plants with PRSV BR, PRSV HA or PRSV TH, Brazilian, Hawaiian and Thai isolates, respectively, revealed lines with mono-, double-, and triple-resistance. After molecular analysis and a preliminary agronomic evaluation, 13 R1 and R2 populations were incorporated into the papaya-breeding program at Embrapa Cassava and Tropical Fruits, in Cruz das Almas, Bahia, Brazil.

Line 63-1: A New Virus-resistant Transgenic Papaya

The disease resistance of a transgenic line expressing the coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) from Hawaii was further analyzed against PRSV isolates from Hawaii and other geographical regions. Line 63-1 originated from the same transformation experiment that resulted in line 55-1 from which the transgenic commercial cultivars, `Rainbow' and `SunUp', were derived. Plants of line 63-1 used in this study consisted of a population from a self pollinated R0 bisexual plant. ELISA and PCR tests provided evidence that there are at least two segregating CP loci. To allow for comparison with reactions of the previously reported line 55-1, virus isolates from Hawaii, Brazil, Thailand, and Jamaica were used to challenge seedlings of 63-1. Unlike line 55-1, a significant percentage of inoculated transgenic plants were susceptible to isolates from Hawaii. However, a proportion of plants were resistant to the non-Hawaiian isolates. In contrast, previous work showed that all plants of the hemizygous line 55-1 were susceptible to PRSV isolates from Brazil, Thailand, and Jamaica. Line 63-1, therefore, presents Hawaii with PRSV-resistant transgenic germplasm that could be used as a source of transgenes for resistance to PRSV isolates within and outside of Hawaii.

Identification and Molecular Characterization of Viruses Infecting Cucurbits in Pakistan

AbstractCucurbits are grown throughout the North‐West Frontier Province of Pakistan as summer and winter crops. Plants having mosaic, mottling, chlorosis and leaf distortion symptoms were frequently found in most of the cucurbit fields during the survey. Using dot immunobinding assay, Cucumber green mottle mosaic virus (CGMMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Papaya ringspot virus (PRSV) were found infecting cucurbits. CGMMV was widespread, infecting 46.9% of the samples tested followed by ZYMV (14.8%), WMV (12.5%) and PRSV (7.8%). Multiple infections were common with 42% of the samples being infected with two viruses and 8% with three viruses. The nucleotide sequences of the coat protein (CP) genes of these four viruses were determined and deduced amino acid sequence comparisons revealed 88.3–99% similarity of the ZYMV‐Pak isolate with other isolates of ZYMV reported worldwide. The amino acid sequence identity of Pakistani isolates of WMV, CGMMV and PRSV ranged from 96.8 to 98.4%, 98.1 to 99.4% and 79.3 to 84.2%, respectively, with other isolates reported elsewhere. Little variability was observed in the sequences of WMV and CGMMV. ZYMV‐Pak was very close to the USA isolate, and the PRSV‐Pak isolate was close to Indian isolates of PRSV possibly reflecting the geographical relationship between these isolates.

Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India.

The coat protein (CP) sequences of eleven Papaya ringspot virus (PRSV) isolates originating from different locations in India were determined, analysed and compared with the sequences of other isolates of PRSV. The virus isolates from India exhibited considerable heterogeneity in the CP sequences. The CP-coding region varied in size from 840-858 nucleotides, encoding protein of 280-286 amino acids. Comparative sequence analysis revealed that the PRSV isolates originating from India were divergent up to 11%. Though the PRSV isolates were differentiated in to two clusters, yet the sequence variation could not be correlated with the geographical origin of the isolates. Implication of the sequence variation in the coat protein derived transgenic resistance in papaya is discussed.

Engineered Resistance Against Papaya ringspot virus in Venezuelan Transgenic Papayas.

Local varieties of papaya grown in the Andean foothills of Mérida, Venezuela, were transformed independently with the coat protein (CP) gene from two different geographical Papaya ringspot virus (PRSV) isolates, designated VE and LA, via Agrobacterium tumefaciens. The CP genes of both PRSV isolates show 92 and 96% nucleotide and amino acid sequence similarity, respectively. Four PRSV-resistant R0 plants were intercrossed or selfed, and the progenies were tested for resistance against the homologous isolates VE and LA, and the heterologous isolates HA (Hawaii) and TH (Thailand) in greenhouse conditions. Resistance was affected by sequence similarity between the transgenes and the challenge viruses: resistance values were higher for plants challenged with the homologous isolates (92 to 100% similarity) than with the Hawaiian (94% similarity) and, lastly, Thailand isolates (88 to 89% similarity). Our results show that PRSV CP gene effectively protects local varieties of papaya against homologous and heterologous isolates of PRSV.

COMPARISON OF THE COAT PROTEIN OF A SOUTH INDIAN STRAIN OF PRSV WITH OTHER STRAINS FROM DIFFERENT GEOGRAPHICAL LOCATIONS

Papaya ring spot virus (PRSV) causes major diseases in papaya and cucurbits in the Indian sub-continent that result in significant yield losses. Molecular characterization of the coat protein (CP) gene of a South Indian strain (INP-UAS) of PRSV-P revealed an open reading frame of 849 bp that encoded the putative coat protein of 283 amino acids (GenBank Acc No. AF528190). The DAG triplet associated with aphid transmissibility and the potential protease cleavage site Q/S, located in the N-terminus of the INP-UAS CP, were conserved, as has been reported for other PRSV coat proteins. The sequence had a deletion of 24 nucleotides that corresponded to eight amino acids in the N-terminal region of the CP. A comparison of the amino acid sequence of the INP-UAS CP with those reported for other PRSV isolates showed that the N-termini were variable and suggested that the distinctiveness of INP-UAS was linked to its geographical location. Phylogenetic analysis also showed that the INP-UAS strain coat protein gene was relatively divergent from those of other PRSV-P isolates as it formed a separate and distinct group. The implications of sequence variability for the use of CPgenes in the development of transgenic plants for viral resistance are discussed.

First Report of Occurrence of Papaya ring spot virus Infecting Papaya in Bangladesh.

Papaya (Carica papaya L.) is an important fruit crop in Bangladesh. During surveys conducted in Dhaka and Mymensingh regions from April to June 2003, >50% of papaya plants were observed to have leaf mottling, mosaic and mild distortion, and water-soaked streaks on petioles and stem, which are typical symptoms of Papaya ring spot virus (PRSV) infection. Electron-microscopic examination of negatively stained leaf-dip preparations from 10 symptomatic samples revealed the association of flexuous virus particles that were decorated with polyclonal antibodies raised to an isolate from India (PRSV-D). The identity of PRSV associated with the papaya disease in Bangladesh was further confirmed by reverse transcription polymerase chain reaction and sequence analysis (2). By using PRSV specific primers (2), the 3'-terminal region comprising a part of the nuclear inclusion b gene, the coat protein (CP) gene, and the untranslated region were amplified and sequenced (GenBank Accession No. AY423557). The CP gene consisted of 286 amino acids and the conserved regions common to the genus Potyvirus, such as WCIEN and QMKAA, were present. Like all known PRSV sequences (1), a stretch of glutamic acid and lysine repeats (EK region) after the aphid transmission motif (DAG) also was present. Comparative CP amino acid sequence analyses revealed that the virus infecting papaya in Bangladesh, designated as PRSV-Bd, shared 89 to 92% identity with PRSV isolates from India and 88 to 93% identity with isolates from other parts of the world. To our knowledge, this is the first report of occurrence of PRSV infecting papaya in Bangladesh.

Broad-Spectrum Resistance to Different Geographic Strains ofPapaya ringspot virusin Coat Protein Gene Transgenic Papaya

ABSTRACT Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.