Development and evaluation of a duplex TaqMan qPCR assay for detection and quantification of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts.

BACKGROUND: A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

The Genomic Landscape of Centromeres in Cancers.

Centromere genomics remain poorly characterized in cancer, due to technologic limitations in sequencing and bioinformatics methodologies that make high-resolution delineation of centromeric loci difficult to achieve. We here leverage a highly specific and targeted rapid PCR methodology to quantitatively assess the genomic landscape of centromeres in cancer cell lines and primary tissue. PCR-based profiling of centromeres revealed widespread heterogeneity of centromeric and pericentromeric sequences in cancer cells and tissues as compared to healthy counterparts. Quantitative reductions in centromeric core and pericentromeric markers (α-satellite units and HERV-K copies) were observed in neoplastic samples as compared to healthy counterparts. Subsequent phylogenetic analysis of a pericentromeric endogenous retrovirus amplified by PCR revealed possible gene conversion events occurring at numerous pericentromeric loci in the setting of malignancy. Our findings collectively represent a more comprehensive evaluation of centromere genetics in the setting of malignancy, providing valuable insight into the evolution and reshuffling of centromeric sequences in cancer development and progression.

Lack of satellite DNA species-specific homogenization and relationship to chromosomal rearrangements in monitor lizards (Varanidae, Squamata).

BACKGROUND: Satellite DNAs (stDNAs) are highly repeated sequences that constitute large portions of any genome. The evolutionary dynamics of stDNA (e.g. copy number, nucleotide sequence, location) can, therefore, provide an insight into genome organization and evolution. We investigated the evolutionary origin of VSAREP stDNA in 17 monitor lizards (seven Asian, five Australian, and five African) at molecular and cytogenetic level. RESULTS: Results revealed that VSAREP is conserved in the genome of Asian and Australian varanids, but not in African varanids, suggesting that these sequences are either differentiated or lost in the African varanids. Phylogenetic and arrangement network analyses revealed the existence of at least four VSAREP subfamilies. The similarity of each sequence unit within the same VSAREP subfamily from different species was higher than those of other VSAREP subfamilies belonging to the same species. Additionally, all VSAREP subfamilies isolated from the three Australian species (Varanus rosenbergi, V. gouldii, and V. acanthurus) were co-localized near the centromeric or pericentromeric regions of the macrochromosomes, except for chromosomes 3 and 4 in each Australian varanid. However, their chromosomal arrangements were different among species. CONCLUSIONS: The VSAREP stDNA family lack homogenized species-specific nucleotide positions in varanid lineage. Most VSAREP sequences were shared among varanids within the four VSAREP subfamilies. This suggests that nucleotide substitutions in each varanid species accumulated more slowly than homogenization rates in each VSAREP subfamily, resulting in non-species-specific evolution of stDNA profiles. Moreover, changes in location of VSAREP stDNA in each Australian varanid suggests a correlation with chromosomal rearrangements, leading to karyotypic differences among these species.

Identification of a Nanovirus-Alphasatellite Complex in Sophora alopecuroides.

Viruses in the genus Nanovirus of the family Nanoviridae generally have eight individually encapsidated circular genome components and have been predominantly found infecting Fabaceae plants in Europe, Australia, Africa and Asia. For over a decade Sophora alopecuroides L. (Fabaceae) plants have been observed across Iran displaying dwarfing, yellowing, stunted leaves and yellow vein banding. Using a high-throughput sequencing approach, sequences were identified within one such plant that had similarities to nanovirus genome components. From this plant, the nanovirus-like molecules DNA-R (n=4), DNA-C (n=2), DNA-S (n=1), DNA-M (n=1), DNA-N (n=1), DNA-U1 (n=1), DNA-U2 (n=1) and DNA-U4 (n=1) were amplified, cloned and sequenced. Other than for the DNA-R, these components share less than 71% identity with those of other known nanoviruses. The four DNA-R molecules were highly diverse, sharing only 65-71% identity with each other and 64-86% identity with those of other nanoviruses. In the S. alopecuroides plant 14 molecules sharing 57.7-84.6% identity with previously determined sequences of nanovirus-associated alphasatellites were also identified. Given the research activity in the nanovirus field during the last five years coupled with high-throughput sequence technologies, many more diverse nanoviruses and nanovirus-associated satellites are likely to be identified.

Frequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan.

Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production on the Indian subcontinent, and is caused by monopartite begomoviruses accompanied by a specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Since the breakdown of resistance against CLCuD in 2001/2002, only one virus, the "Burewala" strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bur), and a recombinant form of CLCuMB have consistently been identified in cotton across the major cotton growing areas of Pakistan. Unusually a bipartite isolate of the begomovirus Tomato leaf curl virus was identified in CLCuD-affected cotton recently. In the study described here we isolated the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) from CLCuD-affected cotton. To assess the frequency and geographic occurrence of ToLCNDV in cotton, CLCuD-symptomatic cotton plants were collected from across the Punjab and Sindh provinces between 2013 and 2015. Analysis of the plants by diagnostic PCR showed the presence of CLCuKoV-Bur in all 31 plants examined and ToLCNDV in 20 of the samples. Additionally, a quantitative real-time PCR analysis of the levels of the two viruses in co-infected plants suggests that coinfection of ToLCNDV with the CLCuKoV-Bur/CLCuMB complex leads to an increase in the levels of CLCuMB, which encodes the major pathogenicity (symptom) determinant of the complex. The significance of these results are discussed.

High genetic homogeneity points to a single introduction event responsible for invasion of Cotton leaf curl Multan virus and its associated betasatellite into China.

BACKGROUND: Cotton leaf curl Multan virus (CLCuMuV) is a Whitefly Transmitted Geminivirus (WTG) endemic to the India subcontinent and is notorious as a causal agent of cotton leaf curl disease (CLCuD), a major constraint to cotton production in south Asia. We found CLCuMuV infecting Hibiscus rosa-sinensis in Guangzhou, China in 2006. The spread and evolution of the invading CLCuMuV were monitored in the following nine years. FINDINGS: CLCuMuV spread rapidly in the last nine years and became established in Southern China. It infects at least five malvaceous plant species, H. rosa-sinensis, H. esculentus, Malvaiscus arboreus, Gossypium hirsutum and H. cannabinus. Complete nucleotide sequences of 34 geographically and/or temporally distinct CLCuMuV isolates were determined and analyzed together with six other publicly available genomes of CLCuMuV occurring in China. The 40 CLCuMuV isolates were found to share > 99 % nucleotide sequence identity with each other. In all cases tested, the CLCuMuVs were associated with a CLCuMuB. The 36 CLCuMuBs (30 sequenced by us) shared > 98 % nucleotide sequence identity. CONCLUSION: The high genetic homogeneity of CLCuMuV and CLCuMuB in China suggests the establishment of them from a single founder event.

Organization and evolution of Gorilla centromeric DNA from old strategies to new approaches.

The centromere/kinetochore interaction is responsible for the pairing and segregation of replicated chromosomes in eukaryotes. Centromere DNA is portrayed as scarcely conserved, repetitive in nature, quickly evolving and protein-binding competent. Among primates, the major class of centromeric DNA is the pancentromeric α-satellite, made of arrays of 171 bp monomers, repeated in a head-to-tail pattern. α-satellite sequences can either form tandem heterogeneous monomeric arrays or assemble in higher-order repeats (HORs). Gorilla centromere DNA has barely been characterized, and data are mainly based on hybridizations of human alphoid sequences. We isolated and finely characterized gorilla α-satellite sequences and revealed relevant structure and chromosomal distribution similarities with other great apes as well as gorilla-specific features, such as the uniquely octameric structure of the suprachromosomal family-2 (SF2). We demonstrated for the first time the orthologous localization of alphoid suprachromosomal families-1 and -2 (SF1 and SF2) between human and gorilla in contrast to chimpanzee centromeres. Finally, the discovery of a new 189 bp monomer type in gorilla centromeres unravels clues to the role of the centromere protein B, paving the way to solve the significance of the centromere DNA's essential repetitive nature in association with its function and the peculiar evolution of the α-satellite sequence.

A novel satellite DNA sequence in the Peromyscus genome (PMSat): Evolution via copy number fluctuation.

Satellite DNAs (satDNA) are tandemly arrayed repeated sequences largely present in eukaryotic genomes, which play important roles in genome evolution and function, and therefore, their analysis is vital. Here, we describe the isolation of a novel satellite DNA family (PMSat) from the rodent Peromyscus eremicus (Cricetidae, Rodentia), which is located in pericentromeric regions and exhibits a typical satellite DNA genome organization. Orthologous PMSat sequences were isolated and characterized from three species belonging to Cricetidae: Cricetus cricetus, Phodopus sungorus and Microtus arvalis. In these species, PMSat is highly conserved, with the absence of fixed species-specific mutations. Strikingly, different numbers of copies of this sequence were found among the species, suggesting evolution by copy number fluctuation. Repeat units of PMSat were also found in the Peromyscus maniculatus bairdii BioProject, but our results suggest that these repeat units are from genome regions outside the pericentromere. The remarkably high evolutionary sequence conservation along with the preservation of a few numbers of copies of this sequence in the analyzed genomes may suggest functional significance but a different sequence nature/organization. Our data highlight that repeats are difficult to analyze due to the limited tools available to dissect genomes and the fact that assemblies do not cover regions of constitutive heterochromatin.

Higher-order repeat structure in alpha satellite DNA occurs in New World monkeys and is not confined to hominoids.

Centromeres usually contain large amounts of tandem repeat DNA. Alpha satellite DNA (AS) is the most abundant tandem repeat DNA found in the centromeres of simian primates. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, which are tandem arrays of larger repeat units consisting of multiple basic repeat units. HOR-carrying AS also occurs in other hominoids, but results reported to date for phylogenetically more remote taxa have been negative. Here we show direct evidence for clear HOR structures in AS of the owl monkey and common marmoset. These monkeys are New World monkey species that are located phylogenetically outside of hominoids. It is currently postulated that the presence of HOR structures in AS is unique to hominoids. Our results suggest that this view must be modified. A plausible explanation is that generation of HOR structures is a general event that occurs occasionally or frequently in primate centromeres, and that, in humans, HOR-carrying AS became predominant in the central region of the centromere. It is often difficult to assemble sequence reads of tandem repeat DNAs into accurate contig sequences; our careful sequencing strategy allowed us to overcome this problem.

Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

Circomics of Cuban geminiviruses reveals the first alpha-satellite DNA in the Caribbean.

Circomics (circular DNA genomics), the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analysis and pyro-sequencing, has been used recently to identify geminiviruses with high efficiency and low costs. Circular DNAs associated with Cuban geminiviruses were characterised by RCA/RFLP analysis and 454 sequencing of two batches of DNA amplified from selected plant samples as well as individual cloning and Sanger sequencing of DNA components and compared to other geminiviral DNAs by phylogenetic analysis. Cuban geminiviruses that were closely related to each other challenged the circomics approach. Ten geminiviral components and one alpha-satellite DNA were determined and compared to three geminiviral components obtained by conventional cloning. New strains of Sida yellow mottle virus (SiYMoV), tomato yellow distortion leaf virus (ToYDLV), Sida golden mosaic Florida virus (SiGMFV) and Sida golden mosaic Liguanea virus (SiGMLV) are described with host plant species being classified by molecular PCR-based bar coding. A new virus species is named Peristrophe mosaic virus. The first alpha-satellite found in Middle America establishes the New World branch of these elements which are related to nanoviruses and were previously thought to be restricted to the Old World. In conclusion, circomics is efficient for complex infections and closely related viruses to detected unexpected viral DNAs, but may need some scrutinisation by direct sequencing and cloning of individual components for certain cases.

Regional changes in the sequence of cotton leaf curl multan betasatellite.

Cotton leaf curl disease (CLCuD) in Pakistan and northwestern India is caused by monopartite begomoviruses in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Following a recent upsurge in CLCuD problems in Sindh province (southern Pakistan), sequences of clones of CLCuMB were obtained from Sindh and Punjab province (central Pakistan), where CLCuD has been a problem since the mid-1980s. The sequences were compared to all sequences of CLCuMB available in the databases. Analysis of the sequences shows extensive sequence variation in CLCuMB, most likely resulting from recombination. The range of sequence variants differ between Sindh, the Punjab and northwestern India. The possible significance of the findings with respect to movement of the CLCuD between the three regions is discussed. Additionally, the lack of sequence variation within the only coding sequence of CLCuMB suggests that the betasatellite is not involved in resistance breaking which became a problem after 2001 in the Punjab and subsequently also in northwestern India.

Association of an alphasatellite with tomato yellow leaf curl virus and ageratum yellow vein virus in Japan is suggestive of a recent introduction.

Samples were collected in 2011 from tomato plants exhibiting typical tomato leaf curl disease symptoms in the vicinity of Komae, Japan. PCR mediated amplification, cloning and sequencing of all begomovirus components from two plants from different fields showed the plants to be infected by Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein virus (AYVV). Both viruses have previously been shown to be present in Japan, although this is the first identification of AYVV on mainland Japan; the virus previously having been shown to be present on the Okinawa Islands. The plant harboring AYVV was also shown to contain the betasatellite Tomato leaf curl Java betasatellite (ToLCJaB), a satellite not previously shown to be present in Japan. No betasatellite was associated with the TYLCV infected tomato plants analyzed here, consistent with earlier findings for this virus in Japan. Surprisingly both plants were also found to harbor an alphasatellite; no alphasatellites having previously been reported from Japan. The alphasatellite associated with both viruses was shown to be Sida yellow vein China alphasatellite which has previously only been identified in the Yunnan Province of China and Nepal. The results suggest that further begomoviruses, and their associated satellites, are being introduced to Japan. The significance of these findings is discussed.

Isolation and characterization of two satellite DNAs in some Iberian rock lizards (Squamata, Lacertidae).

Satellite DNAs represent a large portion of all high eukaryotic genomes. They consist of numerous very similar repeated sequences, tandemly arranged in large clusters up to 100 million base pairs in length, usually located in the heterochromatic parts of chromosomes. The biological significance of satDNAs is still under discussion, but most of their proposed functions are related to heterochromatin and/or centromere formation and function. Because information about the structure of reptilian satDNA is far from exhaustive, we present a molecular and cytogenetic characterization of two satDNA families in four lacertid species. Two families of tandemly repeated DNAs, namely TaqI and HindIII satDNAs, have been cloned and sequenced from four species belonging to the genus Iberolacerta. These satDNAs are characterized by a monomer length of 171-188 and 170-172 bp, and by an AT content of 60.5% and 58.1%, respectively. FISH experiments with TaqI satDNA probe produced bright signals in pericentromeric regions of a subset of chromosomes whereas all the centromeres were marked by HindIII probe. The results obtained in this study suggest that chromosome location and abundance of satDNAs influence the evolution of these elements, with centromeric families evolving tenfold faster than interstitial/pericentromeric ones. Such different rates render different satellites useful for phylogenetic investigation at different taxonomic ranks.

Identification and diversity of functional centromere satellites in the wild rice species Oryza brachyantha.

The centromere is a key chromosomal component for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, allowing each sister chromatid to faithfully segregate to each daughter cell during cell division. It is not clear what types of sequences act as functional centromeres and how centromere sequences are organized in Oryza brachyantha, an FF genome species. In this study, we found that the three classes of centromere-specific CentO-F satellites (CentO-F1, CentO-F2, and CentOF3) in O. brachyantha share no homology with the CentO satellites in Oryza sativa. The three classes of CentO-F satellites are all located within the chromosomal regions to which the spindle fibers attach and are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons, CRR-F, in the O. brachyantha centromeres. Although these CentO-F satellites are quantitatively variable among 12 O. brachyantha centromeres, immunostaining with an antibody specific to CENH3 indicates that they are colocated with CENH3 in functional centromere regions. Our results demonstrate that the three classes of CentO-F satellites may be the major components of functional centromeres in O. brachyantha.

Cotton leaf curl Gezira alphasatellite associated with tomato leaf curl Sudan virus approaches the expected upper size limit in the evolution of alphasatellites.

Begomoviruses (family Geminiviridae) cause severe damage to tomato crops worldwide. Among them, tomato leaf curl disease (ToLCD)-associated begomoviruses are a major concern for tomato production in Sudan. Here, we report the detection of unexpectedly large cotton leaf curl Gezira alphasatellite molecules (up to 1467 nt) associated with an isolate of a novel strain of tomato leaf curl Sudan virus (ToLCSDV) in tomato plants affected by ToLCD. A recombinant nature is suggested for this ToLCSDV isolate.

Genetic diversity and distribution of a distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman.

Tomato and pepper are widely grown in Oman for local consumption. A countrywide survey was conducted during 2010-2011 to collect samples and assess the diversity of begomoviruses associated with leaf curl disease of tomato and pepper. A virus previously only identified on the Indian subcontinent, chili leaf curl virus (ChLCV), was found associated with tomato and pepper diseases in all vegetable grown areas of Oman. Some of the infected plant samples were also found to contain a betasatellite. A total of 19 potentially full-length begomovirus and eight betasatellite clones were sequenced. The begomovirus clones showed >96% nucleotide sequence identity, showing them to represent a single species. Comparisons to sequences available in the databases showed the highest levels of nucleotide sequence identity (88.0-91.1%) to isolates of the "Pakistan" strain of ChLCV (ChLCV-PK), indicating the virus from Oman to be a distinct strain, for which the name Oman strain (ChLCV-OM) is proposed. An analysis for recombination showed ChLCV-OM likely to have originated by recombination between ChLCV-PK (the major parent), pepper leaf curl Lahore virus and a third strain of ChLCV. The betasatellite sequences obtained were shown to have high levels of identity to isolates of tomato leaf curl betasatellite (ToLCB) previous shown to be present in Oman. For the disease in tomato Koch's postulates were satisfied by Agrobacterium-mediated inoculation of virus and betasatellites clones. This showed the symptoms induced by the virus in the presence of the betasatellite to be enhanced, although viral DNA levels were not affected. ChLCV-OM is the fourth begomovirus identified in tomato in Oman and the first in Capsicum. The significance of these findings is discussed.

Molecular characterization of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar.

Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name "Cassava mosaic alphasatellite".

First report of an alphasatellite associated with Okra enation leaf curl virus.

An alphasatellite DNA associated with Okra enation leaf curl virus (OELCuV) which causes enation and leaf curling in okra (Abelmoschus esculentus) plants was characterized. The full-length DNA comprises 1,350 nucleotides and shows typical genome organization of an alphasatellite. It shows the highest nucleotide sequence identity (79.7 %) to Hollyhock yellow vein virus-associated symptomless alphasatellite (HoYVSLA). This is the first report of the association of an alphasatellite with OELCuV from India.

Molecular characterization of begomoviruses and DNA satellites associated with okra leaf curl disease in Cameroon.

Okra leaf curl disease (OLCD) is the most important viral disease of okra in West Africa. In this study, a complex of begomoviruses and associated DNA satellites were identified in symptomatic okra plants from southwestern Cameroon. Sequence analyses showed that two of the plants (Lik1 and Njo5) were infected with a begomovirus being a recombinant of cotton leaf curl Gezira virus (CLCuGeV) and okra yellow crinkle virus (OYCrV). The recombinant genome shared highest nucleotide identity with isolates of CLCuGeV at 87.8% and is therefore considered to be member of a new begomovirus species, Okra leaf curl Cameroon virus (OLCuCMV). One plant (Mue5) was infected by a begomovirus with 95.8% nucleotide identy to CLCuGeV, while in the plants Lik1, Mue1 and Njo5, a begomovirus was identified showing highest nucleotide identity at 93.7% with OYCrV. The nucleotide comparisons and phylogenetic analyses suggest that these isolates represent new Cameroonian strains of CLCuGeV and OYCrV (CLCuGeV-CM and OYCrV-CM). Mixed infection of OLCuCMV and OYCrV-CM was found in two of the plants. A betasatellite and two divergent alphasatellites were also associated with the begomoviruses. The betasatellite was identified as cotton leaf curl Gezira betasatellite (CLCuGeB) with the highest nucleotide identity at 93.3% to other African isolates of CLCuGeB. The alphasatellites, herein named Alpha-1 and Alpha-2, shared 97.3% and 95.2% identity, respectively, with cotton leaf curl Gezira alphasatellite (CLCuGeA) and okra leaf curl Burkina Faso alphasatellite (OLCuBFA). These collective results emphasize the extent of diversity among okra-infecting begomovirus-satellite complexes in western Africa.