MOLECULAR ANALYSES OF PAPAYA VIRUSES IN BANGLADESH: DETECTION, CHARACTERIZATION, AND DISTRIBUTION

Abstract

Aphid-transmitted PRSV is the greatest disease threat to commercial papaya production worldwide. Specific, ultrasensitive assays are important for the early detection of PRSV in the field. I developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of the virus. The concentrations and annealing temperatures of both primer pairs were optimized to avoid potential competition between primer sets during STNP. The assay was more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. RT-PCR and ELISA were capable of detecting PRSV 14 to 21 days post-inoculation, whereas STNP detected PRSV in plants 7d post-inoculation. This new STNP assay also detected PRSV from virus-infected asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV titers in the field and in imported plant samples. As in other countries, PRSV is the major limitation to papaya production in Bangladesh. Full-length coding genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse transcriptase polymerase chain reaction (RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74% and 83% identical to each other at the nucleotide and amino acid levels, respectively. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical to other full-length PRSV sequences at the nucleotide level, respectively. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a distinct branch from the other PRSV sequences by nucleotide as well as amino acid sequence comparisons. Comparisons of the genome sequences of these newly identified PRSV strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment with the highest identity to PRSV-Meghalaya (MF356497) and the other contained a 5,105-nucleotide fragment with the highest identity to PRSV-China (KY933061). The occurrence of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh was approximately 19% and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The new full-length genome sequences of these PRSV strains from Bangladesh and their distribution provide important information on the dynamics of papaya ringspot virus infections in papaya in Bangladesh. Forty-five papaya samples with severe leaf curl symptoms were tested by PCR using a degenerate primer set for virus species in the genus Begomovirus. Of these, 29 were positive for tomato leaf curl Bangladesh virus (ToLCBV). The complete genome sequences of ToLCBV (GenBank accession no. MH380003) and its associated tomato leaf curl betasatellite (ToLCB) (MH397223) from papaya isolate Gaz17-Pap were determined and characterized. Defective betasatellites were found in ToLCBV-positive papaya isolates Gaz19-Pap, Gaz20-Pap and Gaz21-Pap. This study confirmed that papaya is a host of ToLCBV, ToLCB, and other defective and recombinant DNA satellites in Bangladesh. The complete genome sequence of tomato leaf curl Joydebpur virus (ToLCJoV) and its associated tomato leaf curl Joydebpur betasatellite (ToLCJoB) were determined and characterized from papaya isolate J1-Pap. ToLCJoV infecting papaya was most closely related to ToCJoV reported from Gazipur, Bangladesh, causing tomato leaf curl disease of tomato. ToLCB infecting papaya had the highest homologies at nucleotide and amino acid levels to ToLCB from tomato in India. The complete genome sequences of the DNA A and DNA B components of tomato leaf curl New Delhi virus (ToLCNDV) were determined and characterized from papaya isolate ND-71. ToLCNDV infecting papaya was most closely related to ToLCNDV reported from Gazipur, Bangladesh, causing tomato leaf curl disease of tomato. DNA B of ToLCNDV infecting papaya had the highest homologies at nucleotide and amino acid levels of DNA B of ToLCNDV from tomato in India.