Mosaic Recombination Research Articles

Mosaic Recombination Inflicted Various SARS-CoV-2 Lineages to Emerge into Novel Virus Variants: a Review Update.

Human Coronaviruses (hCoVs) belongs to the enormous and dissimilar family of positive-sense, non-segmented, single-stranded RNA viruses. The RNA viruses are prone to high rates of mutational recombination resulting in emergence of evolutionary variant to alter various features including transmissibility and severity. The evolutionary changes affect the immune escape and reduce effectiveness of diagnostic and therapeutic measures by becoming undetectable by the currently available diagnostics and refractory to therapeutics and vaccines. Whole genome sequencing studies from various countries have adequately reported mosaic recombination between different lineage strain of SARS-CoV-2 whereby RNA dependent RNA polymerase (RdRp) gene reconnects with a homologous RNA strand at diverse position. This all lead to evolutionary emergence of new variant/ lineage as evident with the emergence of XBB in India at the time of writing this review. The continuous periodical genomic surveillance is utmost required for understanding the various lineages involved in recombination to emerge into hybrid variant. This may further help in assessing virus transmission dynamics, virulence and severity factor to help health authorities take appropriate timely action for prevention and control of any future COVID-19 outbreak.

Characterization of Near Full-Length Transmitted/Founder HIV-1 Subtype D and A/D Recombinant Genomes in a Heterosexual Ugandan Population (2006-2011).

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5′ and 3′ half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

Characterization of multi-cellular dynamics of angiogenesis and vascular remodelling by intravital imaging of the wounded mouse cornea

Establishment of the functional blood vasculature involves extensive cellular rearrangement controlled by growth factors, chemokines and flow-mediated shear forces. To record these highly dynamic processes in mammalians has been technically demanding. Here we apply confocal and wide field time-lapse in vivo microscopy to characterize the remodelling vasculature of the wounded mouse cornea. Using mouse lines with constitutive or inducible endogenous fluorescent reporters, in combination with tracer injections and mosaic genetic recombination, we follow processes of sprouting angiogenesis, sprout fusion, vessel expansion and pruning in vivo, at subcellular resolution. We describe the migratory behaviour of endothelial cells of perfused vessels, in relation to blood flow directionality and vessel identity. Live-imaging following intravascular injection of fluorescent tracers, allowed for recording of VEGFA-induced permeability. Altogether, live-imaging of the remodelling vasculature of inflamed corneas of mice carrying endogenous fluorescent reporters and conditional alleles, constitutes a powerful platform for investigation of cellular behaviour and vessel function.

HIV-1 transmission among injection drug users leads to novel recombinants circulating in southern China.

Injection drug use has been responsible for most HIV-1 transmission in Asia and has led to the generation and/or spreading of circulating recombinant CRF01_AE, CRF07_BC, and CRF08_BC, which are currently the predominant HIV-1 strains in China. Monitoring HIV-1 transmissions among injection drug users (IDUs) still provides critical information on HIV-1 prevalence and control. In the present study, we detected three HIV-1 sequences, BH048/057/066, from IDUs in Beihai city, Guangxi province, all of which share an extremely complex mosaic recombination pattern. CRF01_AE and CRF08_BC contributed to the rise of new recombinants, which contain CRF08_BC-derived env fragments with evidence of further evolution. Our data, combined with those of other studies, suggest that the BH048/057/066 recombinants represent early strains of a circulating recombinant form of HIV-1 in southern China. Information concerning such newly appearing variants is critical for both HIV-1 control and ongoing HIV-1 vaccine trials in Guangxi, China.

Loss of functional Dicer in mouse radial glia cell-autonomously prolongs cortical neurogenesis

Radial glia of the mouse cerebral cortex emerge from neuroepithelial stem cells around embryonic day 11 and produce excitatory cortical neurons until a few days before birth. The molecular mechanisms that regulate the end of cortical neurogenesis remain largely unknown. Here we investigated if the Dicer-dependent microRNA (miRNA) pathway is involved. By electroporating a cre-recombinase expression vector into the cortex of E13.5 embryos carrying a conditional allele of Dicer1, we induced mosaic recombination causing Dicer1 deletion and reporter activation in a subset of radial glia. We analysed the long-term fates of their progeny. We found that mutant radial glia produced abnormally large numbers of Cux1-positive neurons, many of which populated the superficial cortical layers. Injections of the S-phase marker bromodeoxyuridine between postnatal days 3 and 14 showed that much of this population was generated postnatally. Our findings suggest a role for Dicer-dependent processes in limiting the timespan of cortical neurogenesis.

Postnatal Expression of BRAFV600E Does Not Induce Thyroid Cancer in Mouse Models of Thyroid Papillary Carcinoma

The mutant BRAF (BRAF(V600E)) is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The oncogenicity of this mutation has been shown by some genetically engineered mouse models. However, in these mice, BRAF(V600E) is expressed in all the thyroid cells from the fetal periods, and suppresses thyroid function, thereby leading to TSH elevation, which by itself promotes thyroid tumorigenesis. To overcome these problems, we exploited 2 different approaches, both of which allowed temporally and spatially restricted expression of BRAF(V600E) in the thyroid glands. First, we generated conditional transgenic mice harboring the loxP-neo(R)-loxP-BRAF(V600E)-internal ribosome entry site-green fluorescent protein sequence [Tg(LNL-BRAF(V600E))]. The double transgenic mice (LNL-BRAF(V600E);TPO-Cre) were derived from a high expressor line of Tg(LNL-BRAF(V600E)) mice and TPO-Cre mice; the latter expresses Cre DNA recombinase under the control of thyroid-specific thyroid peroxidase (TPO) promoter and developed PTC-like lesions in early life under normal serum TSH levels due to mosaic recombination. In contrast, injection of adenovirus expressing Cre under the control of another thyroid-specific thyroglobulin (Tg) promoter (Ad-TgP-Cre) into the thyroids of LNL-BRAF(V600E) mice did not induce tumor formation despite detection of BRAF(V600E) and pERK in a small fraction of thyroid cells. Second, postnatal expression of BRAF(V600E) in a small number of thyroid cells was also achieved by injecting the lentivirus expressing loxP-green fluorescent protein-loxP-BRAF(V600E) into the thyroids of TPO-Cre mice; however, no tumor development was again observed. These results suggest that BRAF(V600E) does not appear to induce PTC-like lesions when expressed in a fraction of thyroid cells postnatally under normal TSH concentrations.

Divergent roles for Wnt/β-catenin signaling in epithelial maintenance and breakdown during semicircular canal formation

The morphogenetic program that shapes the three semicircular canals (SSCs) must be executed with extreme precision to satisfy their complex vestibular function. The SSCs emerge from epithelial outgrowths of the dorsal otocyst, the central regions of which fuse and resorb to leave three fluid-filled canals. The Wnt/β-catenin signaling pathway is active at multiple stages of otic development, including during vestibular morphogenesis. How Wnt/β-catenin functionally integrates with other signaling pathways to sculpt the SSCs and their sensory patches is unknown. We used a genetic strategy to spatiotemporally modulate canonical Wnt signaling activity during SSC development in mice. Our findings demonstrate that Wnt/β-catenin signaling functions in a multifaceted manner during SSC formation. In the early phase, Wnt/β-catenin signaling is required to preserve the epithelial integrity of the vertical canal pouch perimeter (presumptive anterior and posterior SSCs) by establishing a sensory-dependent signaling relay that maintains expression of Dlx5 and opposes expression of the fusion plate marker netrin 1. Without this Wnt signaling activity the sensory to non-sensory signaling cascade fails to be activated, resulting in loss of vestibular hair and support cells and the anterior and posterior SSCs. In the later phase, Wnt/β-catenin signaling becomes restricted to the fusion plate where it facilitates the timely resorption of this tissue. Mosaic recombination of β-catenin in small clusters of canal pouch cells prevents their resorption, causing instead the formation of ectopic SSCs. Together, these disparate functions of the Wnt/β-catenin pathway in epithelial maintenance and resorption help regulate the size, shape and number of SSCs.

Use of PB-Cre4 Mice for Mosaic Gene Deletion

Transgene expression from short promoters in transgenic animals can lead to unwanted transgene expression patterns, often as a byproduct of random integration of the expression cassette into the host genome. Here I demonstrate that the often used PB-Cre4 line (also referred to as “Probasin-Cre”), although expressing exclusively in the male prostate epithelium when transmitted through male mice, can lead to recombination of loxP-flanked alleles in a large variety of tissues when transmitted through female mice. This aberrant Cre activity due to Cre expression in the oocytes leads to different outcomes for maternally or paternally transmitted loxP-flanked alleles: Maternally inherited loxP-flanked alleles undergo recombination very efficiently, making female PB-Cre4 mice an efficient monoallelic “Cre deleter line”. However, paternally inherited loxP-flanked alleles are inefficiently recombined by maternal PB-Cre4, giving rise to mosaic expression patterns in the offspring. This mosaic recombination is difficult to detect with standard genotyping approaches of many mouse lines and should therefore caution researchers using PB-Cre4 to use additional approaches to exclude the presence of recombined alleles. However, mosaic recombination should also be useful in transgenic “knockout” approaches for mosaic gene deletion experiments.

HIV-1 Subtypes and Recombinants in Northern Tanzania: Distribution of Viral Quasispecies

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28–50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies.

In vivo evaluation of PhiC31 recombinase activity in transgenic mice

Genome engineering strategies employing site-specific recombinases (SSRs) such as Cre, Flp and PhiC31, have become powerful tools to analyze gene function and manipulate neural network in vertebrates. In the present study, we evaluated the ability of PhiC31 phage integrase to induce genomic recombination in transgenic mice. PhiC31 is the integrase encoded by the Streptomyces bacteriophage that promotes recombination between heterotypic attP and attB sites. We generated transgenic mice that express codon-optimized PhiC31 (PhiC31o) in neural stem/progenitor cells or tyrosine hydroxylase (TH) expressing catecholaminergic neurons. PhiC31 was functional in these cells and capable of excising a transcriptional stop cassette flanked by PhiC31-specific attP/B recognition sites. PhiC31-ERT2, a fusion protein of PhiC31o (without the nuclear localization signal) and the mutated ligand-binding domain of the human estrogen receptor, was able to induce recombination in neural stem/progenitor cells in a tamoxifen-dependent manner, but the recombination rate was less efficient than for PhiC31. Thus, PhiC31 integrase is functional in transgenic mice and is suitable for mosaic recombination in restricted cell populations.

Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA.

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.

Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.

Inheritance of DNA Transferred from American Trypanosomes to Human Hosts

Interspecies DNA transfer is a major biological process leading to the accumulation of mutations inherited by sexual reproduction among eukaryotes. Lateral DNA transfer events and their inheritance has been challenging to document. In this study we modified a thermal asymmetric interlaced PCR by using additional targeted primers, along with Southern blots, fluorescence techniques, and bioinformatics, to identify lateral DNA transfer events from parasite to host. Instances of naturally occurring human infections by Trypanosoma cruzi are documented, where mitochondrial minicircles integrated mainly into retrotransposable LINE-1 of various chromosomes. The founders of five families show minicircle integrations that were transferred vertically to their progeny. Microhomology end-joining of 6 to 22 AC-rich nucleotide repeats in the minicircles and host DNA mediates foreign DNA integration. Heterogeneous minicircle sequences were distributed randomly among families, with diversity increasing due to subsequent rearrangement of inserted fragments. Mosaic recombination and hitchhiking on retrotransposition events to different loci were more prevalent in germ line as compared to somatic cells. Potential new genes, pseudogenes, and knockouts were identified. A pathway of minicircle integration and maintenance in the host genome is suggested. Thus, infection by T. cruzi has the unexpected consequence of increasing human genetic diversity, and Chagas disease may be a fortuitous share of negative selection. This demonstration of contemporary transfer of eukaryotic DNA to the human genome and its subsequent inheritance by descendants introduces a significant change in the scientific concept of evolutionary biology and medicine.

Placenta‐specific gene activation and inactivation using integrase‐defective lentiviral vectors with the Cre/LoxP system

Transgenic and knockout studies have advanced our understanding of the genetic control of embryonic development over the past decades. However, interpretation of the phenotype of mutant mice is potentially complicated, since the commonly used knockout approach modifies both the fetal and placental genome. To circumvent this problem, we previously developed a placenta-specific gene manipulation system by lentiviral vector transduction of embryos at the blastocyst stage. In the present study, by combination with the Cre/LoxP system, we successfully demonstrate placenta-specific gene activation and inactivation in EGFP reporter mice and Ets2 floxed mice, respectively. Transient expression using integrase-defective lentiviral (IDLV) vectors diminished the toxic effect of Cre expression and solved the dilemma of mosaic recombination with lower concentrations and toxic effects with higher concentrations of Cre recombinase. We also show that placenta-specific Ets2 disruption causes embryonic lethality and reconfirmed the critical role of Ets2 during placentation. This technology facilitates both gain and loss of gene function analyses in placental development during pregnancy. Since IDLV vectors can efficiently transduce a variety of cell types similarly to wild-type vectors, our IDLV-Cre strategy is potentially useful for a wide range of applications.

Novel HIV-1 long terminal repeat (LTR) sequences of subtype B and mosaic intersubtype B/C recombinants in North India

Although the HIV-1 epidemic in India is mainly due to subtype C, other subtypes have also been reported from different parts of India. HIV-1 LTR sequence analysis from six HIV-1 infected individuals from North India was carried out to determine the nature and extent of variations. Four out of six samples formed a unique phylogenetic cluster which was close to subtype B. The other two samples (A3 and S3) turned out to be novel mosaic recombinants showing resemblance to subtypes B, B/C-India and B/C-Myanmar gene segments. All four subtype B LTR samples and the two B/C recombinants showed conserved as well as unique polymorphisms in all of the putative transcription factor binding sites (TFBS). These changes may potentially alter basal as well as Tat-mediated HIV-1 LTR promoter activation. The two recombinants possessed three copies of the NF-kappaB TFBS as seen with the majority of subtype C and recombinant B/C isolates reported earlier, but the other four non-recombinant B-LTRs showed only two copies of the NF-kappaB site. This is the first study to show a dominance of unique subtype B-LTRs and strongly suggests that this region could also be a hot spot for the formation of highly complex inter subtype B/C recombinants.

The incomplete inactivation ofFgf8 in the limb ectoderm affects the morphogenesis of the anterior autopod through BMP-mediated cell death

Here we analyze limb development after the conditional inactivation of Fgf8 from the epiblast, using the previously described MORE (Mox2Cre) line. This line drives variable mosaic recombination of a floxed Fgf8 allele, resulting in a small proportion of AER cells that maintain Fgf8 expression. The phenotype of Mox2Cre;Fgf8 limbs is most similar to that of Msx2Cre;Fgf8 forelimbs, indicating that a small but durable expression of FGF8 is equivalent to an early normal, but transitory, expression. This functional equivalence likely relies on the subsequent Fgf4 upregulation that buffers the differences in the pattern of Fgf8 expression between the two conditional mutants. The molecular analysis of Mox2Cre;Fgf8 limbs shows that, despite Fgf4 upregulation, they develop under reduced FGF signaling. These limbs also exhibit an abnormal area of cell death at the anterior forelimb autopod, overlapping with an ectopic domain of Bmp7 expression, which can explain the abnormal morphogenesis of the anterior autopod.

An exact nonparametric method for inferring mosaic structure in sequence triplets.

Statistical tests for detecting mosaic structure or recombination among nucleotide sequences usually rely on identifying a pattern or a signal that would be unlikely to appear under clonal reproduction. Dozens of such tests have been described, but many are hampered by long running times, confounding of selection and recombination, and/or inability to isolate the mosaic-producing event. We introduce a test that is exact, nonparametric, rapidly computable, free of the infinite-sites assumption, able to distinguish between recombination and variation in mutation/fixation rates, and able to identify the breakpoints and sequences involved in the mosaic-producing event. Our test considers three sequences at a time: two parent sequences that may have recombined, with one or two breakpoints, to form the third sequence (the child sequence). Excess similarity of the child sequence to a candidate recombinant of the parents is a sign of recombination; we take the maximum value of this excess similarity as our test statistic Delta(m,n,b). We present a method for rapidly calculating the distribution of Delta(m,n,b) and demonstrate that it has comparable power to and a much improved running time over previous methods, especially in detecting recombination in large data sets.

Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues

Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-Ras(V12) mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.

High-efficiency CAG-FLPe Deleter Mice in C57BL/6J Background

The increasing popularity of conditional knockout (KO) technology has resulted in the demand for efficient FLP deleter mice. In addition, FLP deleters are needed in genetic backgrounds that are suited to behavioral studies. We generated CAG-FLPe transgenic (Tg) mice with the C57BL/6J genetic background, which is one of the most commonly-used strains in behavioral studies. We assessed the recombination efficiency of the CAG-FLPe-Tg lines by crossing them with a mouse line carrying a FRT-PGK-neo-FRT cassette. Four of five independent CAG-FLPe lines induced recombination in most (91%-100%) of their progenies, although a small fraction (0%-30%, depending on the line) showed mosaic recombination patterns. These animals are highly potent as deleters of FRT cassettes and are useful for behavioral studies involving conditional KO mice.

Recombination in circulating Human enterovirus B: independent evolution of structural and non-structural genome regions

The complete nucleotide sequences of eight Human enterovirus B (HEV-B) strains were determined, representing five serotypes, E6, E7, E11, CVB3 and CVB5, which were isolated in the former Soviet Union between 1998 and 2002. All strains were mosaic recombinants and only the VP2-VP3-VP1 genome region was similar to that of the corresponding prototype HEV-B strains. In seven of the eight strains studied, the 2C-3D genome region was most similar to the prototype E30, EV74 and EV75 strains, whilst the remaining strain was most similar to the prototype E1 and E9 strains in the non-structural protein genome region. Most viruses also bore marks of additional recombination events in this part of the genome. In the 5' non-translated region, all strains were more similar to the prototype E9 than to other enteroviruses. In most cases, recombination mapped to the VP4 and 2ABC genome regions. This, together with the star-like topology of the phylogenetic trees for these genome regions, identified these genome parts as recombination hot spots. These findings further support the concept of independent evolution of enterovirus genome fragments and indicate a requirement for more advanced typing approaches. A range of available phylogenetic methods was also compared for efficient detection of recombination in enteroviruses.