The Role of Satellites in the Evolution of Begomoviruses.

Begomoviruses have emerged as destructive pathogens of crops, particularly in the tropics and subtropics, causing enormous economic losses and threatening food security. Epidemics caused by begomoviruses have even spread in regions and crops that were previously free from these viruses. The most seriously affected crops include cassava; cotton; grain legumes; and cucurbitaceous, malvaceous, and solanaceous vegetables. Alphasatellites, betasatellites, and deltasatellites are associated with the diseases caused by begomoviruses, but begomovirus-betasatellite complexes have played significant roles in the evolution of begomoviruses, causing widespread epidemics in many economically important crops throughout the world. This article provides an overview of the evolution, distribution, and approaches used by betasatellites in the suppression of host plant defense responses and increasing disease severity.

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses.

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

Association of a begomovirus-satellite complex with yellow vein and leaf curl disease of hollyhock (Alcea rosea) in India.

Geminiviruses cause considerable yield loss in several crop plants worldwide. In 2016, several hollyhock plants displaying yellow mosaic and leaf curling symptoms were noticed in a nursery of Jawaharlal Nehru University, New Delhi, India. Analysis of the collected samples indicated an association of monopartite and bipartite begomoviruses with satellites. Three begomoviruses (including a member of a new begomovirus species), two alphasatellites, and a betasatellite were isolated from yellow-mosaic-disease-affected plants. Similarly, a begomovirus, two alphasatellites, and a betasatellite were found to be associated with leaf curl disease of hollyhock. These begomoviruses and satellites were found to be recombinants. By harboring diverse begomoviruses and satellite DNAs, hollyhock may serve as a potential source of virus inoculum.

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

"Rhizomania" of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus (genus Necrovirus; family Tombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman.

BACKGROUND: In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. METHODS: Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. RESULTS: The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. CONCLUSIONS: The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

ICTV virus taxonomy profile: Sarthroviridae.

The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

Complete nucleotide sequence and genome organization of red clover associated virus 1 (RCaV1), a putative member of the genus Waikavirus (family Secoviridae, order Picornavirales).

Using high-throughput sequencing, a novel waikavirus was identified in a mixed virus infection of red clover (Trifolium pratense L.). Its complete genomic sequence was determined and characterized. The virus, tentatively named red clover associated virus 1 (RCaV1), is phylogenetically related to members of the genus Waikavirus (family Secoviridae, order Picornavirales).

Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites.

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Two distinct begomoviruses associated with an alphasatellite coinfecting Emilia sonchifolia in Thailand.

Emilia sonchifolia is a traditionally used medicinal plant that is widespread in tropical and subtropical regions of the world. Yellow vein symptoms were observed in E. sonchifolia plants in fields in the county of Koh Samui, Surat Thani Province, Thailand, in August 2015. Two distinct begomoviruses, designated TH4872-6 and TH4872-9, and an associated alphasatellite were obtained from an E. sonchifolia leaf sample (TH4872). Sequence analysis showed that the full-length sequence of TH4872-6 was most closely related to that of ageratum yellow vein China virus (AYVCNV), with 85.7% identity, suggesting that it is a novel begomovirus, while the TH4872-9 sequence closely resembled cotton leaf curl Multan virus (CLCuMuV) with 99.1% identity. The alphasatellite sequence showed the highest nucleotide sequence identity (92.8%) to an isolate of tobacco curly shoot alphasatellite (TbCSA) originating from China. Recombination analysis revealed that the isolate TH4872-6 is a potential recombinant begomovirus, derived from ageratum yellow vein virus (AYVV) and tobacco leaf curl Thailand virus (TbLCTHV). This study represents the first report of begomoviruses identified in E. sonchifolia in Thailand.

Characterization of the complete genome of euonymus yellow vein associated virus, a distinct member of the genus Potexvirus, family Alphaflexiviridae, isolated from Euonymus bungeanus Maxim in Liaoning, Northern China.

In August 2016, a yellow vein disease was observed on leaves of Euonymus bungeanus Maxim (Euonymus, Celastraceae) in Liaoning, China. Virions measuring 750 × 13 nm were observed in a sample from the diseased plant. A potexvirus was detected in the sample by small-RNA deep sequencing analysis and recovered by traditional cloning. The genome of this potexvirus consists of 7,279 nucleotides, excluding the poly(A) tail at the 3' end, and contains five open reading frames (ORFs). Based on the nucleotide and amino acid sequences of the coat protein gene, the virus shared the highest sequence similarity with white clover mosaic virus (WCMV, X16636) (40.1%) and clover yellow mosaic virus (ClYMV, D00485) (37.1%). Phylogenetic analysis showed that the virus clustered with potexviruses and is most closely related to strawberry mild yellow edge virus. These results indicate that this virus is a distinct member of the genus Potexvirus, for which the name euonymus yellow vein associated virus (EuYVAV) is proposed. To our knowledge, this is the first report of a potexvirus on E. bungeanus.

A neo-virus lifestyle exhibited by a (+)ssRNA virus hosted in an unrelated dsRNA virus: Taxonomic and evolutionary considerations.

Recent studies illustrate that fungi as virus hosts provides a unique platform for hunting viruses and exploring virus/virus and virus/host interactions. Such studies have revealed a number of as-yet-unreported viruses and virus/virus interactions. Among them is a unique intimate relationship between a (+)ssRNA virus, yado-kari virus (YkV1) and an unrelated dsRNA virus, yado-nushi virus (YnV1). YkV1 dsRNA, a replicated form of YkV1, and RNA-dependent RNA polymerase, are trans-encapsidated by the capsid protein of YnV1. While YnV1 can complete its replication cycle, YkV1 relies on YnV1 for its viability. We previously proposed a model in which YkV1 diverts YnV1 capsids as the replication sites. YkV1 is neither satellite virus nor satellite RNA, because YkV1 appears to encode functional RdRp and enhances YnV1 accumulation. This represents a unique mutualistic virus/virus interplay and similar relations in other virus/host fungus systems are detectable. We propose to establish the family Yadokariviridae that accommodates YkV1 and recently discovered viruses phylogenetically related to YkV1. This article overviews what is known and unknown about the YkV1/YnV1 interactions. Also discussed are the YnV1 Phytoreo_S7 and YkV1 2A-like domains that may have been captured via horizontal transfer during the course of evolution and are conserved across extant diverse RNA viruses. Lastly, evolutionary scenarios are envisioned for YkV1 and YnV1.

A novel East African monopartite begomovirus-betasatellite complex that infects Vernonia amygdalina.

The complete genomes of a monopartite begomovirus (genus Begomovirus, family Geminiviridae) and an associated betasatellite found infecting Vernonia amygdalina Delile (family Compositae) in Uganda were cloned and sequenced. Begomoviruses isolated from two samples showed the highest nucleotide sequence identity (73.1% and 73.2%) to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus, and betasatellites from the same samples exhibited the highest nucleotide sequence identity (67.1% and 68.2%) to vernonia yellow vein Fujian betasatellite. Following the current taxonomic criteria for begomovirus species demarcation, the isolates sequenced here represent a novel begomovirus species. Based on symptoms observed in the field, we propose the name vernonia crinkle virus (VeCrV) for this novel begomovirus and vernonia crinkle betasatellite (VeCrB) for the associated betasatellite. This is the first report of a monopartite begomovirus-betasatellite complex from Uganda.

A classification system for virophages and satellite viruses.

Satellite viruses encode structural proteins required for the formation of infectious particles but depend on helper viruses for completing their replication cycles. Because of this unique property, satellite viruses that infect plants, arthropods, or mammals, as well as the more recently discovered satellite-like viruses that infect protists (virophages), have been grouped with other, so-called "sub-viral agents." For the most part, satellite viruses are therefore not classified. We argue that possession of a coat-protein-encoding gene and the ability to form virions are the defining features of a bona fide virus. Accordingly, all satellite viruses and virophages should be consistently classified within appropriate taxa. We propose to create four new genera - Albetovirus, Aumaivirus, Papanivirus, and Virtovirus - for positive-sense single-stranded (+) RNA satellite viruses that infect plants and the family Sarthroviridae, including the genus Macronovirus, for (+)RNA satellite viruses that infect arthopods. For double-stranded DNA virophages, we propose to establish the family Lavidaviridae, including two genera, Sputnikvirus and Mavirus.

Monitoring the dynamics of emergence of a non-canonical recombinant of Tomato yellow leaf curl virus and displacement of its parental viruses in tomato.

Recombinant viruses are increasingly being reported but the dynamics of their emergence is rarely documented. A new recombinant Tomato yellow leaf curl virus (TYLCV-IS76) was detected for the first time in 2010 in Southern Morocco (Souss). An original diagnostic tool was needed to fit its unusual recombination profile. Although IS76 was detected following the appearance of Tylc symptoms on tolerant tomato plants, symptoms could not be associated to IS76 or to a synergy with criniviruses. According to infection profiles of Tylc-associated viruses determined on 879 plant samples collected between 1998 and 2014 and a Bayesian inference applied to genomic sequences of representatives of TYLCV, IS76 emerged in Southern Morocco at the end of the 1990s, replaced the parental viruses between 2004 and 2012 in Souss and is spreading towards the North of Morocco. The emergence of IS76 coincides with the increasing use of tolerant cultivars in the 2000s.

Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India.

Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report on a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60 × 10- 3 substitutions site- 1 year- 1) and the ßC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57 × 10- 4 substitution site- 1 year- 1; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22 × 10- 4 substitution site- 1 year- 1]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.

High-level diversity of tailed phages, eukaryote-associated viruses, and virophage-like elements in the metaviromes of antarctic soils.

The metaviromes of two distinct Antarctic hyperarid desert soil communities have been characterized. Hypolithic communities, cyanobacterium-dominated assemblages situated on the ventral surfaces of quartz pebbles embedded in the desert pavement, showed higher virus diversity than surface soils, which correlated with previous bacterial community studies. Prokaryotic viruses (i.e., phages) represented the largest viral component (particularly Mycobacterium phages) in both habitats, with an identical hierarchical sequence abundance of families of tailed phages (Siphoviridae > Myoviridae > Podoviridae). No archaeal viruses were found. Unexpectedly, cyanophages were poorly represented in both metaviromes and were phylogenetically distant from currently characterized cyanophages. Putative phage genomes were assembled and showed a high level of unaffiliated genes, mostly from hypolithic viruses. Moreover, unusual gene arrangements in which eukaryotic and prokaryotic virus-derived genes were found within identical genome segments were observed. Phycodnaviridae and Mimiviridae viruses were the second-most-abundant taxa and more numerous within open soil. Novel virophage-like sequences (within the Sputnik clade) were identified. These findings highlight high-level virus diversity and novel species discovery potential within Antarctic hyperarid soils and may serve as a starting point for future studies targeting specific viral groups.

Mixed infection by two West African tomato-infecting begomoviruses and ageratum leaf curl Cameroon betasatellite in tomato in Cameroon.

Begomovirus isolates ToF3B2 and ToF3B17 and betasatellite isolate SatBToF3 were obtained from the same infected tomato plant showing begomovirus disease symptoms in Fontem, Cameroon. The full-length nucleotide sequences of ToF3B2, ToF3B17 and SatBToF3 were cloned and sequenced and were determined to be 2,797 nt, 2,794 and 1,373 nt long respectively. When compared with other begomovirus and betasatellite sequences, ToF3B2 was 93.5 % identical to Tomato leaf curl Togo virus, ToF3B17 was 95 % identical to Tomato leaf curl Cameroon virus and SatBToF3 was 92 % identical to Ageratum leaf curl Cameroon betasatellite (ALCCMB), respectively. The identification of ALCCMB in Ageratum and now in tomato strongly suggests Ageratum may be an alternative host to these viruses and that ALCCMB is non host specific and may cause severe diseases when transmitted to other crops.

Isolation and molecular characterization of a distinct begomovirus and its associated betasatellite infecting Hedyotis uncinella (Hook. et Arn.) in Vietnam.

A begomovirus isolate VN1 associated with symptomatic Hedyotis uncinella Hook. et Arn. from Vietnam was characterized. The virus, which we provisionally name H. uncinella yellow mosaic virus (HUYMV), has a monopartite genome of 2,749 nucleotides (nts). Pairwise comparisons of DNA-A sequences showed that HUYMV had a maximum nt sequence identity with Soybean crinkle leaf virus (SbLCV) and Premna leaf curl virus (PLCuV) at 82.1 and 81.9 %, respectively, which are less than the 89 % identity in the complete genome, which has been used as the threshold value for demarcation of species in the genus Begomovirus, the family Geminiviridae. One recombination event was detected for HUYMV, which involves an unknown begomovirus as the major parent and Tomato leaf curl Philippines virus (ToLCPV) as the minor parent, with nt 2163 and nt 2452 as the beginning and ending breakpoints, respectively. A betasatellite was found to be associated with HUYMV. The betasatellite showed the highest nt sequence identity (70 %) with Tomato leaf curl Philippine betasatellite--[Philippines:Laguna2:2006]. The name H. uncinella yellow mosaic betasatellite [Vietnam: Binh Dinh: 2013] was proposed for the betasatellite.

Real-time PCR systems targeting giant viruses of amoebae and their virophages.

Giant viruses that infect amoebae, including mimiviruses and marseilleviruses, were first described in 2003. Virophages were subsequently described that infect mimiviruses. Culture isolation with Acanthamoeba spp. and metagenomic studies have shown that these giant viruses are common inhabitants of our biosphere and have enabled the recent detection of these viruses in human samples. However, the genomes of these viruses display substantial genetic diversity, making it a challenge to examine their presence in environmental and clinical samples using conventional and real-time PCR. We designed and evaluated the performance of PCR systems capable of detecting all currently isolated mimiviruses, marseilleviruses and virophages to assess their prevalence in various samples. Our real-time PCR assays accurately detected all or most of the members of the currently delineated lineages of giant viruses infecting acanthamoebae as well as the mimivirus virophages, and enabled accurate classification of the mimiviruses of amoebae in lineages A, B or C. We were able to detect four new mimiviruses directly from environmental samples and correctly classified these viruses within mimivirus lineage C. This was subsequently confirmed by culture on amoebae followed by partial Sanger sequencing. PCR systems such as those implemented here may contribute to an improved understanding of the prevalence of mimiviruses, their virophages and marseilleviruses in humans.