Recombination Detection Program Research Articles

Recombination study using Radish leaf curl virus isolates

Begomovirus isolates, exhibiting identity of 89 and 93% with Radish leaf curl virus (RaLCV), were detected from the leaf curl disease affected okra (Abelmoschus esculentus) plants in Bihar, India. The detected isolates are supposed to be a new strain of RaLCV and therefore are referred to here as RaLCV-Hajipur1 and RaLCV-Hajipur2. Alphasatellite was identified as Cotton leaf curl Burewala alphasatellite (CLCuBuA) and betasatellite as Tomato leaf curl Bangladesh betasatellite (ToLCBDB). The aforementioned RaLCV-Hajipur isolates were used to construct infectious clones. These isolates were inoculated separately or together with alpha- and betasatellites in tobacco. The begomovirus and satellites were isolated from tobacco plants at twenty seven day post inoculation and then sequenced. Analysis of the sequences revealed the presence of a new isolate, referred to here as RaLCV-Hajipur3. Recombination analysis using recombination detection program 3 (RDP3) revealed that the RaLCVHajipur1, RaLCV-Hajipur2 and RaLCV-Hajipur3 had recombinant origin. RDP3 analysis of the RaLCVHajipur3 revealed that the two inoculated isolates (RaLCV-Hajipur1 and RaLCV-Hajipur2) were the probable major and minor parents of the newly detected isolate (RaLCV-Hajipur3).

Evidence for different patterns of natural inter-genotype recombination between two PCV2 parental strains in the field

Co-infection with different virus strains is a precondition for genome recombination, which give rise to continuous evolution of porcine circovirus type 2 (PCV2). In the present study, 32 PCV2 positive clinical samples from the diseased and dead pigs were identified by classic PCR. 15 of 32 (46.8%) clinical samples were identified as infection with both PCV2a and PCV2b using genotype-specific PCR. 13/15 of PCV2 strains were identified as recombinants using sequencing analysis, phylogenetic analysis, recombination detection program and base-by-base comparison. Further analyses of the full-length sequences of these strains suggest that the natural recombination events occurred between strains DQ104423 (PCV2a) and AY579893 (PCV2b), yield two new recombinant clusters through different recombination patterns with crossover regions located in ORF2. Recombinant cluster 1 included 3 strains, and recombinant cluster 2 included 10 strains. These results demonstrate that recombination between PCV2a and PCV2b strains can occur in cap protein coding region through different patterns and yield different recombinants. Our study not only provided new evidences that PCV2 strains can undergo recombination through a variety of patterns, but also suggests that recombination events easily occur in the co-existence of different strains of PCV2.

Two natural recombinant highly pathogenic porcine reproductive and respiratory syndrome viruses with different pathogenicities

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a variant of type 2 PRRSV with high virulence. Genetic and pathogenic characteristics of HP-PRRSV vary rapidly during the evolution. In this study, we determined the complete genome of a HP-PRRSV isolate designated 10FUJ-2, which shared 98.34 % nucleotide identity with HP-PRRSV reference strain JXA1. Genomic analyses by phylogenetic tree and recombination detection program confirmed 10FUJ-2 to be a recombinant with 09JS and JXA1 as potential parental viruses. Furthermore, we identified that 10FUJ-2 has high virulence as similar as the parental viruses by animal challenge study. In addition, we found that SY0909 was also a recombination virus probably from JXA1 and NT0801, which has been reported to be low pathogenic. Recombination analysis also revealed that Glycoproteins GP2 to GP5 of HP-PRRSV might contain major virulence determinants. Identification of two natural recombinants with different virulence supports the notion that recombination is a driving force affecting HP-PRRSV pathogenicity and a common mechanism contributing to HP-PRRSV evolution.

Analysis of HIV-1 gp120 quasispecies suggests high prevalence of intra-subtype recombination

Recombination between viruses of the same HIV-1 subtype has been understudied primarily due to the lack of reference sequences, as well as appropriate bio-informatics tools. The introduction of recombination detection program (RDP3) made the detection of HIV-1 intra-subtype recombination feasible. Methods This study determined the prevalence and patterns of HIV-1 intra-subtype recombinants among female bar and hotel workers in Moshi, Tanzania. The HIV-1 env gp120 V1-C5 quasispecies from 45 subjects classified as pure HIV-1 subtypes A1, C, or D were analyzed for recombination events by RDP3. Results HIV-1 quasispecies with evidence for recombination were found in 89% of subjects infected with pure HIV-1 subtypes A, C, and D. Recombinant viruses were observed at both the baseline and the 12 month visits in 88% of the subjects; in 12% of subjects recombination was identified only at the later time point. Two major patterns were observed: 70% of subjects had unique recombination breakpoints without dominance of any particular variant, while in 30% of subjects a specific recombinant variant dominated in the pool of viral quasispecies. Conclusion A large proportion of female bar and hotel workers were infected with intra-subtype recombinant viruses. These results suggest that HIV-1 co- and super-infections are common in this population, and occur more frequently than previously thought. Intra-subtype recombination contributes to the increased viral diversity which poses a challenge to HIV-1 vaccine design.

Recombination and natural selection in hepatitis E virus genotypes

To gain new insights into the evolutionary processes that created the genetic diversity of the hepatitis E virus (HEV), the Recombination Detection Program (RDP) and SimPlot program were employed to detect recombination events in the genome, then the fixed-effects likelihood (FEL) method was used to detect natural selection effects on viral proteins. Recombination analysis provided strong evidence for both intergenotype and intragenotype recombination events in the sequences analyzed. Recombination events were found to be distributed non-randomly, with the highest frequency in the X domain and the helicase. Strain DQ450072 was identified as intergenotype-recombinant. Natural selection analysis revealed that codons under both negative selection and positive selection were distributed non-randomly. ORF1 and ORF2 have experienced strong purifying selection across genotypes. Furthermore, potentially important sites were also found under positive selection in the N-terminal end of ORF2 and the C-terminal end of ORF3. No significant difference was found among the selective pressures on different genotypes.

Detection of natural inter- and intra-genotype recombination events revealed by cap gene analysis and decreasing prevalence of PCV2 in wild boars

Porcine circovirus type 2 (PCV2), the causative agent of a number of PCVAD (porcine circovirus associated diseases), is ubiquitous in domestic pig and wild boar populations. In the present study, using recombination detection program, phylogenetic analysis and base-by-base comparison of 28 PCV2 ORF2s (capsid protein coding gene) from wild boars and 8 from domestic pigs of Transylvania, recent natural intra- (PCV2b-1B/PCV2b-1C) and inter-genotype (PCV2a-2D/PCV2b-1C) recombination events were detected. Notably, one potential recombinant (F1-21) was detected in domestic pig with possible parental strains of wild boar origin. The estimated recombinant breakpoints comprised epitopes A, B and C of ORF2, without major changes in amino acid sequences. The prevalence of PCV2 in the wild boar population during the 5-year period following the first outbreaks of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs in Romania showed a decrease from 13.4% to 8.3%. To our knowledge, this is the first study to show the existence of ORF2-based intra- and inter-genotype recombination in wild boar populations and the possible recombination between PCV2 strains of wild boars and domestic pigs. Our results suggest a certain independence of PCV2 infection in wild boar populations and demonstrate the possibility of infection with multiple PCV2 genotypes under natural circumstances. On the other hand, PCV2 genotypes specific for wild boars could be detected in domestic pig at lower frequency suggesting the possible spread of wild boar PCV2 to domestic swine. The recombination events described here may contribute to the genetic diversity of PCV2 and may also be the source of emergence of new PCV2 strains.

Molecular Evidence of Recombination on Korean Isolates of Tomato yellow leaf curl virus by Nucleotide Transversions and Transitions

Tomato yellow leaf curl virus (TYLCV), a member of genus Begomovirus, was isolated in Korea in 2008. We sequenced and analyzed the DNA-A of 51 TYLCV isolates from Korea, and 13 of the TYLCV isolates were selected as type representatives of TYLCV from six Korean provinces. The 13 TYLCV isolates were classified into Korea Group 1 (KG1, nine isolates) and Korea Group 2 (KG2, four isolates) based on the results of phylogenetic analysis and genome size (2774 and 2781 nucleotides, respectively). A recombination detection program 3 (RDP3) revealed two recombinations between the TYLCV Korea isolates and other TYLCV isolates [Thailand (AF206674), Iran (AJ132711), and Israel (X76319)]. TYLCV Jeju isolate was characterized by two recombination events (E1 and E2) caused by the presence of E1 in ORF V1 and C3, which may seem to be the mutations of the high nucleotide transversion and transition rate. Collectively, our results suggest that the occurrence of nucleotide transversions and transitions in TYLCV DNA-A might have induced novel recombination events within the TYLCV Korea isolates.

Biological and molecular characterization of a recombinant isolate of Watermelon mosaic virus associated with a watermelon necrotic disease in Italy

The biological and molecular characterization is reported of a Watermelon mosaic virus isolate, denoted WMV-Le, associated with a necrotic phenotype of watermelon plants grown in the Provinces of Lecce and Taranto (Apulia, southern Italy). The fully sequenced WMV-Le genome consists of 10,045 nucleotides and is 99.1% similar to that of WMV-C05-270, a French isolate from melon of the WMV molecular group 3. Using recombination detection program RDP3, putative recombination breakpoints were identified close to nucleotide positions 42 to 1892, covering the 5′UTR/P1/HC-Pro region. The event represents the insertion of a sequence fragment of an isolate similar to WMV-FBR04-37 in the background of an isolate similar to WMV-FMF00-LL1. The field symptomatology was reproduced in watermelon plants grown in an experimental greenhouse but the virus induced severe symptoms also in Cucumis sativus, C. melo, Cucurbita maxima and C. pepo .

Recombination in the Nonstructural Gene Region in Type 2 Dengue Viruses

In view of the rising prevalence of dengue virus and mixing of host and vector populations, complete full-length sequence determination of dengue strains isolated from different epidemic areas is important for the study of virus evolution, pathogenicity, vaccine efficiency and diagnosis. Based on the genomic analysis of 51 complete dengue virus sequences, all of which cocirculated in Thailand between 1974 and 2001, we report here the occurrence of homologous recombination in the NS5 nonstructural gene region of dengue virus type 2 (DENV-2) strains. In order to analyze those 51 virus sequences at one time, we chose to use a highly sensitive recombination detection program called RDP. When RDP detects a possible recombination event, further bootscanning and phylogenetic tree analyses are applied to these candidate sequences to identify this recombination event. We found that within the DENV-2 subfamily, the strain ThNH63/93 is the evolutionary product of a recombination event between ThNH62/93 and ThD2_0284_90 strains. The strain ThNH62/93 was identified as the major parent, while the strain ThD2_0284_90 was the minor parent. The recombination site was determined to localize between positions 7967 (±36 nt) and 8283 (±36 nt) with a significance level of p < 0.001. Our results showed, for the first time, that an intraserotype recombination event occurred between DENV-2 strains in the nonstructural gene region; by contrast, an interserotype recombination between different serotypes of dengue strains was not identified. This study thus supports the theory that homologous recombination plays a key role in dengue virus evolution.

Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence

Human adenovirus type 53 (HAdV-53) has commonly been detected in samples from epidemic keratoconjunctivitis (EKC) patients in Japan since 1996. HAdV-53 is an intermediate virus, containing hexon-chimeric, penton base and fiber structures similar to HAdV-22 and -37, HAdV-37 and HAdV-8, respectively. HAdV-53-like intermediate strains were first isolated from EKC samples in Japan in the 1980s. Here, the complete genome sequences of three such HAdV-53-like intermediate strains (870006C, 880249C and 890357C) and four HAdV-53 strains were determined, and their relationships were analysed. The seven HAdV strains were classified into three groups, 870006C/880249C, 890357C and the four HAdV-53 strains, on the basis of phylogenetic analyses of the partial and complete genome sequences. HAdV strains within the same group showed the highest nucleotide identities (99.87-100.00 %). Like HAdV-53, the hexon loop 1 and 2 regions of 870006C, 880249C and 890357C showed the highest identity with HAdV-22. However, these strains did not show a hexon-chimeric structure similar to HAdV-22 and -37, or a penton base similar to HAdV-37. The fiber genes of 870006C and 880249C were identical to that of HAdV-37, but not HAdV-8. Thus, the three intermediate HAdVs isolated in the 1980s were similar to each other but not to HAdV-53. The recombination breakpoints were inferred by the Recombination Detection Program (rdp) using whole-genome sequences of these seven HAdV and of 12 HAdV-D strains from GenBank. HAdV-53 may have evolved from intermediate HAdVs circulating in the 1980s, and from HAdV-8, -22 and -37, by recombination of sections cut at the putative breakpoints.

Chilli leaf curl Palampur virus is a distinct begomovirus species associated with a betasatellite

Capsicum frutescens (chilli) plants exhibiting leaf curl symptoms, collected from Palampur, Himachal Pradesh region of India, were found associated with a begomovirus and a betasatellite‐like molecule. Viral DNA was amplified, cloned and sequenced. The begomoviral genome and the betasatellite consisted of 2775 and 1376 nucleotides respectively. The virus appeared to be monopartite. The genome sequence had &lt;87·9% identity with all other begomovirus sequences, below the threshold for species demarcation, suggesting that the isolate represents a distinct species for which the name Chilli leaf curl Palampur virus (ChiLCPaV) is proposed. Infectious clones consisting of partial tandem repeats of the viral genome (1·9‐mer) and the betasatellite (1·7‐mer) were constructed in the binary vector pCAMBIA‐1300 and agroinoculated to chilli and Nicotiana benthamiana. The viral clone did not produce leaf curl symptoms when inoculated alone but the plants were stunted compared to controls. Typical leaf curling and stunting symptoms were observed when the viral clone was inoculated with the betasatellite. In a phylogenetic analysis with other closely related begomoviruses, the new sequence grouped with an isolate of Papaya leaf curl virus‐[Pakistan:2008] from Rhynchosia capitata and Tomato leaf curl New Delhi virus‐[India:2006] from tomato. The betasatellite showed most identity (94%) with an isolate of Chilli leaf curl betasatellite from Pakistan. Using the rdp3 recombination detection program, a recombination event involving Croton yellow vein mosaic virus‐[India:2007] as a major parent and Tomato leaf curl Karnataka virus‐[India:2007] as a minor parent was detected in the ChiLCPaV sequence. No significant recombination event was detected in the betasatellite sequence.

Natural recombinants derived from different patterns of recombination between two PCV2b parental strains

Porcine circovirus diseases (PCVD) are globally emerging diseases that have huge economic impacts on swine industry. Porcine circovirus type 2 (PCV2) is considered to be the essential primary causative agent of PCVD. In the present study, recombination analyses of PCV2 identified two possible recombination events with high confidence using recombination detection program, phylogenetic analysis and base-by-base comparison. These recombination events occurred between strains 09CQ (HQ395024) and ZhuJi2003 ( AY579893), giving rise to two recombinants 09GS (HQ395028) and HN0907 (GU938303). Phylogenetic analyses of the parental strains at full length level suggest that natural recombination happened between PCV2b strains. Interestingly, recombination of the two parental strains yielded two recombinants through different recombination patterns with crossover regions mainly located in ORF1 and ORF2, respectively. These results demonstrate that recombination between PCV2b strains can occur both in non-structural protein coding region and structural protein coding region. Our study not only indicates that PCV2b strains can undergo recombination through a variety of patterns, but also suggests that recombination contributes to the genetic diversity of PCV2.

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.

Molecular characterization of a new species of Begomovirus and betasatellite causing leaf curl disease of tomato in India

A new tomato-infecting begomovirus and cognate betasatellite were characterized from the gangetic plain of northern India. Genome organization of this virus was found to be similar to those of other old world begomoviruses. The DNA-A molecule (2752 nt) shared maximum (85.8%) identity with Tomato leaf curl Laos virus-[Laos] (ToLCLV-[LA]; AF195782) from Laos and betasatellite molecule (1349 nt) shared maximum (75.8%) identity with Tomato leaf curl Joydebpur betasatellite (ToLCJoB-[BD:Gaz:05]; AJ966244) from Bangladesh. Interestingly, both these molecules showed less identity with known tomato-infecting begomoviruses and their satellites from India. The recombination detection program (RDP) revealed that these molecules are not an outcome of direct exchange of sequences between existing begomovirus species. According to International Committee on Taxonomy of viruses (ICTV) species/strains demarcations norms for viruses belonging to the family Geminiviridae, this is a new Begomovirus species and we named this virus as Tomato leaf curl Patna virus (ToLCPaV) and new beta species as Tomato leaf curl Patna betasatellite (ToLCPaB). Partial tandem repeats of ToLCPaV and ToLCPaB could induce typical leaf curl symptom on tomato ( Solanum lycopersicum) and Nicotiana benthamiana. Although, DNA-A could alone infect tomato typical to a monopartite Begomovirus, co-inoculation of DNA-A and DNA-β resulted more stunting and severe symptoms. Interestingly, association of ToLCPaB did not assist in increased ToLCPaV accumulation in systemic leaves. ToLCPaV neither transreplicate DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) nor of Tomato leaf curl Gujarat virus (ToLCGV), presumably due to difference in rep-binding sequences. However, ToLCPaB formed viable pseudorecombinant with mono-bipartite ToLCGV DNA-A infecting both N. benthamiana and tomato but could not cause systemic infection on natural host tomato when co-inoculated with ToLCNDV DNA-A, which is a bipartite Begomovirus.

Analysis of the Coat Protein Gene of Indian Strain of Apple Stem Grooving Virus

A survey was undertaken in the temperate fruit growing regions of Himachal Pradesh (HP) and Jammu & Kashmir (J&K). Apple stem grooving virus (ASGV), a Capillovirus, was detected in different cultivars of apple, nectarines, plum, cherry, quince and apricot by double antibody sandwich ELISA (DAS-ELISA). The coat protein (CP) gene sequence of an amplicon produced by RT-PCR, confirmed the association of ASGV in apple cultivar Starkrimson, collected from Himachal Pradesh. The CP of Indian ASGV isolate shared 100 % sequence identity with a Brazilian isolate (AF438409). Sequence analysis by Recombination Detection Program (RDP2) indicated no recombination event for the Indian isolate. However, recombination was detected in Chinese, Korean and Citrus tatter leaf virus-Taiwan (CTLV) strains of ASGV. The study describes first report of ASGV infection in India and characterization of its CP gene.

Sequence characterization of cotton leaf curl virus from Rajasthan: phylogenetic relationship with other members of geminiviruses and detection of recombination

Diseased cotton plants showing typical leaf curl symptoms were collected from experimental plot of Agriculture Research Station-Sriganganagar, Rajasthan. Complete DNA-A component from samples taken from two areas were amplified through rolling circle amplification (RCA) using templiphi kit (GE Healthcare) and characterized. DNA-A of one isolate consists of 2751 nucleotides and second isolate of 2759 nucleotide. Both sequences comprised six ORF's. Genome organization of DNA-A of one isolate shows high sequence similarity with other characterized local begomovirus isolates of Rajasthan, while other isolate shows high sequence similarity with CLCuV reported from Pakistan. The maximum similarity of first isolate, CLCuV-SG01, shows highest sequence identity with Cotton leaf curl Abohar (Rajasthan) virus, and second isolate, CLCuV-SG02, shows highest sequence identity with cotton leaf curl virus from Pakistan. Both isolates showed 85% similarities with each other. The sequence data revealed probable infiltration of some strains of Cotton leaf curl virus from Pakistan to India, or co-existence of different isolates under similar geographical conditions. While CLCuV-SG01 shows highest nt sequence similarity with CLCuV Rajasthan (Abohar), nt identity of V1 ORF (encoding coat protein) of SG01 shows the highest nt identity (100%) with CLCuV Multan (Bhatinda) and Abohar virus while AC1 region also showed difference. Complete nucleotide sequence of SG01 shows only 86% similarity with CLCuV Multan virus. Similarity search revealed significant difference in AV1 and AC1 regions with respect to DNA-A suggesting an evolutionary history of recombination. Computer based analysis, recombination detection Program (RDP) supports the recombination hypothesis, indicated that recombination with other begomoviruses had taken place within V1 ORF and AC1 ORF of CLCuV-SG01 and AC1 ORF of CLCuV-SG02 and also in noncoding intergenic region (IR).

Role of Recombination in the Evolution of Host Specialization Within Bean yellow mosaic virus

Seven complete genomes and 64 coat protein gene sequences belonging to Bean yellow mosaic virus (BYMV) isolates from different continents were examined for evidence of genetic recombination using six different recombination-detection programs. In the seven complete genomes and a single complete genome of the related virus Clover yellow vein virus (ClYVV), evidence for eight recombination patterns was found by four or more programs, giving firm evidence of their presence, and five additional recombination patterns were detected by three or fewer programs, giving tentative evidence of their occurrence. When the nucleotide sequences of 64 BYMV and one ClYVV coat protein genes were analyzed, three firm recombination patterns were detected in 21 isolates (32%). With another six isolates (9%), tentative evidence was found for three further recombination patterns. Of the 19 firm or tentative recombination patterns detected within and between strain groups of BYMV, and with ClYVV, 12 involved a generalist group of isolates as a parent but none of the other BYMV groups acted as parents more than six times. These findings suggest that recombination played an important role in the evolution of BYMV strain groups that specialize in infecting particular groups of domesticated plants.

Molecular characterization of an almond isolate of Prune dwarf virus in Tunisia: putative recombination breakpoints in the partial sequences of the coat protein-encoding gene in isolates from different geographic origin

Recombination is an important driving force in evolution. To investigate this evolutionary process in Prune dwarf virus (PDV), 31 accessions retrieved from international databases, supplemented by one Tunisian isolate described here, were analyzed. Two recombination detection programs were used: RDP v3.31β and RECCO. While the first program did not detect any significant recombination events, RECCO detected several breakpoints in partial sequences of the coat protein gene (CP) of fifteen isolates. The Tajima neutrality test implemented in the MEGA4.1β program indicated that there were numerous deletion/insertion events in the sequences. The strongest signal was found in Portuguese isolate 3.12N.14 (accession number AY646846) which had position nt 296–523 in the CP gene. The evolutionary historical relationships between all analyzed isolates were determined by constructing a dendrogram using Neighbor joining (NJ), Minimum evolution (ME), Maximum parsimony (MP), Maximum likelihood (ML), and the Unweighted pair group method with arithmetic mean (UPGMA); all gave similar results. Two main clusters were delineated, one representing recombinant and one nonrecombinant isolates. The recombinant isolates were mostly collected from Portugal, whereas the nonrecombinant isolates originated in eastern Europe.

Evidence for Recombination Between Feline Panleukopenia Virus and Canine Parvovirus Type 2

Canine parvovirus type 2 (CPV) is a virulent pathogen that emerged in the late 1970s, probably originating from feline panleukopenia virus (FPLV) or a closely related carnivore parvovirus belonging to the feline parvovirus (FPV) subspecies. In contrast to FPLV, CPV has evolved rapidly since its emergence. The original antigenic type of CPV disappeared more than two decades ago and several new antigenic as well as genetic CPV variants have appeared and spread in the field. Both high mutation rate and positive selection of mutations in the capsid gene appear to be the driving force for such rapid evolution. In addition, genetic recombination has been assessed as a factor in parvovirus evolution. Recently, we provided the first evidence of inter-antigenic type recombination of CPV in nature. Here, an inter-FPV subspecies recombinant was revealed by analyzing the genetic data deposited in databases with several recombination detection programs, and by phylogeny. FPLV strain XJ-1, submitted by Su et al., Harbin, China in 2007 (GenBank accession no. EF988660), was most likely generated by recombination between CPV and FPLV. Its genome was generally composed of the NS1 gene of CPV origin and the VP1 gene of FPLV origin. This is the first demonstration of recombination between different FPV subspecies in nature. Consequently, recombination should be considered as an element in the generation and evolution of parvoviruses of the FPV subspecies.

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.