Multiple Recombination Events Research Articles

Characteristics of the mosaic genome of a human parechovirus type 1 strain isolated from an infant with pneumonia in China

Human parechoviruses (HPeVs) belong to the Parechovirus genus of the large and growing family of Picornaviridae with a non-enveloped, single-stranded and positive-sense RNA. An HPeV strain was isolated from the nasopharyngeal aspirate specimen of a 2months old infant hospitalized with pneumonia in Beijing, China and nominated as BJ-37359 followed the code of the specimen. Strain BJ-37359 was identified as HPeV1 by whole genome sequencing. The full genome of strain BJ-37359 consisted of 7336 nucleotides (nt), excluding a poly (A) tail and contained an ORF of 6537 nt flanked by 5′UTR of 709 nt and 3′UTR of 90nt. Phylogenetic analyses revealed that strain BJ-37359 were clustered together with HPeV1 strains in the structural capsid protein region, while uncoupling in the non-structural gene regions. Analyses with Simplot and Bootscan indicated that multiple recombination events occurred in the non-structural region and VP0 region of strain BJ-37359 with other HPeV1, and other types might have contributed to the recombination, especially HPeV6 and HPeV7 strains. Recombination analyses indicated that strain BJ-37359 may have a mosaic genome with new genomic recombination breakpoints.

Genome characterization of a novel chicken picornavirus distantly related to the members of genus Avihepatovirus with a single 2A protein and a megrivirus-like 3′ UTR

The members of the genus Avihepatovirus and related picornaviruses (“Aalivius”) of ducks, turkey and chickens possess identical 2A peptide composition including three functionally unrelated 2A peptides which is a characteristic genome feature of these monophyletic avian picornaviruses. The complete genome of a novel picornavirus provisionally named Orivirus A1 (KM203656) from a cloacal sample of a 4-week-old diarrheic chicken (Gallus gallus domesticus) distantly related to members of genus Avihepatovirus was characterized. The study strain contains a type-II-like IRES, a single 2A protein of unknown function unrelated to the 2A proteins of avihepatoviruses and a long 3′ untranslated region (UTR) with multiple repeated sequence motifs followed by an AUG-rich region. The repeated sequences of the 3′ UTR show significant identity to the “Unit A” sequences of the phylogenetically distant megriviruses. The presence of a novel single 2A and the megrivirus-like “Unit A” motifs suggest multiple recombination events in the evolution of this novel picornavirus.

Cross-border sexual transmission of the newly emerging HIV-1 clade CRF51_01B.

A novel HIV-1 recombinant clade (CRF51_01B) was recently identified among men who have sex with men (MSM) in Singapore. As cases of sexually transmitted HIV-1 infection increase concurrently in two socioeconomically intimate countries such as Malaysia and Singapore, cross transmission of HIV-1 between said countries is highly probable. In order to investigate the timeline for the emergence of HIV-1 CRF51_01B in Singapore and its possible introduction into Malaysia, 595 HIV-positive subjects recruited in Kuala Lumpur from 2008 to 2012 were screened. Phylogenetic relationship of 485 amplified polymerase gene sequences was determined through neighbour-joining method. Next, near-full length sequences were amplified for genomic sequences inferred to be CRF51_01B and subjected to further analysis implemented through Bayesian Markov chain Monte Carlo (MCMC) sampling and maximum likelihood methods. Based on the near full length genomes, two isolates formed a phylogenetic cluster with CRF51_01B sequences of Singapore origin, sharing identical recombination structure. Spatial and temporal information from Bayesian MCMC coalescent and maximum likelihood analysis of the protease, gp120 and gp41 genes suggest that Singapore is probably the country of origin of CRF51_01B (as early as in the mid-1990s) and featured a Malaysian who acquired the infection through heterosexual contact as host for its ancestral lineages. CRF51_01B then spread rapidly among the MSM in Singapore and Malaysia. Although the importation of CRF51_01B from Singapore to Malaysia is supported by coalescence analysis, the narrow timeframe of the transmission event indicates a closely linked epidemic. Discrepancies in the estimated divergence times suggest that CRF51_01B may have arisen through multiple recombination events from more than one parental lineage. We report the cross transmission of a novel CRF51_01B lineage between countries that involved different sexual risk groups. Understanding the cross-border transmission of HIV-1 involving sexual networks is crucial for effective intervention strategies in the region.

Comparative genome analysis identifies few traits unique to the Escherichia coli ST131 H30Rx clade and extensive mosaicism at the capsule locus.

BackgroundE.coli ST131 is a globally disseminated clone of multi-drug resistant E. coli responsible for that vast majority of global extra-intestinal E. coli infections. Recent global genomic epidemiological studies have highlighted the highly clonal nature of this group of bacteria, however there appears to be inconsistency in some phenotypes associated with the clone, in particular capsule types as determined by K-antigen testing both biochemically and by PCR.ResultsWe performed improved quality assemblies on ten ST131 genomes previously sequenced by our group and compared them to a new reference genome sequence JJ1886 to identify the capsule loci across the drug-resistant clone H30Rx. Our data shows considerable genetic diversity within the capsule locus of H30Rx clone strains which is mirrored by classical K antigen testing. The varying capsule locus types appear to be randomly distributed across the H30Rx phylogeny suggesting multiple recombination events at this locus, but that this capsule heterogeneity has little to no effect on virulence associated phenotypes in vitro.ConclusionsOur data provides a framework for determining the capsular genetics of E. coli ST131 and further beyond to ExPEC strains, and highlights how capsular mosaicism may be an important strategy in becoming a successful globally disseminated human pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-830) contains supplementary material, which is available to authorized users.

Complete Genome Characterization of a Novel Enterovirus Type EV-B106 Isolated in China, 2012

Human enterovirus B106 (EV-B106) is a recently identified member of enterovirus species B. In this study, we report the complete genomic characterization of an EV-B106 strain (148/YN/CHN/12) isolated from an acute flaccid paralysis patient in Yunnan Province, China. The new strain had 79.2–81.3% nucleotide and 89.1–94.8% amino acid similarity in the VP1 region with the other two EV-B106 strains from Bolivia and Pakistan. When compared with other EV serotypes, it had the highest (73.3%) VP1 nucleotide similarity with the EV-B77 prototype strain CF496-99. However, when aligned with all EV-B106 and EV-B77 sequences available from the GenBank database, two major frame shifts were observed in the VP1 coding region, which resulted in substantial (20.5%) VP1 amino acid divergence between the two serotypes. Phylogenetic analysis and similarity plot analysis revealed multiple recombination events in the genome of this strain. This is the first report of the complete genome of EV-B106.

Genomic Analyses of DNA Transformation and Penicillin Resistance in Streptococcus pneumoniae Clinical Isolates

Alterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

High Levels of Multiple Infections, Recombination and Horizontal Transmission of Wolbachia in the Andricus mukaigawae (Hymenoptera; Cynipidae) Communities

Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W8, W2 and W6 apparently have derived from W3 and W5a, W6 and W7, W4 and W9, respectively. W8 and W2 and their respective parental strains were detected in S. japonicus. W6 was detected with only one parent (W4) in S. japonicus; W9 was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.

Origin, Evolution, and Genotyping of Emergent Porcine Epidemic Diarrhea Virus Strains in the United States

ABSTRACTCoronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV.

Enterovirus 74 Infection in Children

Enterovirus 74 (EV74) is a rarely detected viral infection of children. In 2010, EV74 was identified in New Zealand in a 2 year old child with acute flaccid paralysis (AFP) through routine polio AFP surveillance. A further three cases of EV74 were identified in children within six months. These cases are the first report of EV74 in New Zealand. In this study we describe the near complete genome sequence of four EV74 isolates from New Zealand, which shows only limited sequence identity in the non-structural proteins when compared to the other two known EV74 sequences. As is typical of enteroviruses multiple recombination events were evident, particularly in the P2 region and P3 regions. This is the first complete EV74 genome sequenced from a patient with acute flaccid paralysis.

Multiple Genomic Recombination Events in the Evolution of Saffold Cardiovirus

BackgroundSaffold cardiovirus (SAFV) is a new human cardiovirus with 11 identified genotypes. Little is known about the natural history and pathogenicity of SAFVs.Methodology/Principal FindingsWe sequenced the genome of five SAFV-1 strains which were identified from fecal samples taken from children with viral diarrhea in Beijing, China between March 2006 and November 2007, and analyzed the phylogenetic and phylodynamic properties of SAFVs using the genome sequences of every known SAFV genotypes. We identified multiple recombination events in our SAFV-1 strains, specifically recombination between SAFV-2, -3, -4, -9, -10 and the prototype SAFV-1 strain in the VP4 region and recombination between SAFV-4, -6, -8, -10, -11 and prototype SAFV-1 in the VP1/2A region. Notably, recombination in the structural gene VP4 is a rare event in Cardiovirus. The ratio of nonsynonymous substitutions to synonymous substitutions indicates a purifying selection of the SAFV genome. Phylogenetic and molecular clock analysis indicates the existence of at least two subclades of SAFV-1 with different origins. Subclade 1 includes two strains isolated from Pakistan, whereas subclade 2 includes the prototype strain and strains isolated in China, Pakistan, and Afghanistan. The most recent common ancestor of all SAFV genotypes dates to the 1710s, and SAFV-1, -2, and -3 to the 1940s, 1950s, and 1960s, respectively. No obvious relationship between variation and pathogenicity exists in the critical domains of the CD and EF loops of viral capsid proteins or the multi-functional proteins L based on animo acid sequence identity comparison between SAFV genotypes.Conclusions/SignificanceOur findings suggest that intertypic recombination plays an important role in the diversity of SAFVs, highlighting the diversity of the five strains with the previously described SAFV-1 strains.

A new genotype in the genus Arthrospira (Oscillatoriales, Cyanobacteria) revealed by a mosaic-like structure of the 16S-23SrRNA intergenic spacer region in strain PCC 9901

Comte K., Coursin T. and Carre-Mlouka A. 2013. A new genotype in the genus Arthrospira (Oscillatoriales, Cyanobacteria) revealed by a mosaic-like structure of the 16S-23SrRNA intergenic spacer region in strain PCC 9901. Phycologia 52: 333–337. DOI: 10.2216/12–063.1We examined the phylogenetic assignment of the Chinese strain PCC 9901, a cyanobacterium previously identified as Lyngbya laxespiralis, using phenotypic and molecular approaches. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain PCC 9901 clustered with Arthrospira rather than Lyngbya. While all the Arthrospira strains were assigned to four genotypes (clusters IA, IB, IIA and IIB) based on the 16S-23S internal transcribed spacer (ITS) rRNA sequences, we reported here an unexpected new genotype for strain PCC 9901 that we named cluster III. A mosaic-like structure was found in two of the three intergenic spacer regions of the ITS (ISR1 and ISR3). In addition, a short repeated sequence was specific for genotype I, and a motif was found in genotype II sequences, but they were absent from the PCC 9901 ITS sequence, suggesting multiple recombination events and/or divergent evolution. Strain PCC 9901 was therefore placed into Arthrospira, and it constituted a new ITS cluster, together with other Asian strains.

Nonrandom Distribution of Interhomolog Recombination Events Induced by Breakage of a Dicentric Chromosome inSaccharomyces cerevisiae

Dicentric chromosomes undergo breakage in mitosis, resulting in chromosome deletions, duplications, and translocations. In this study, we map chromosome break sites of dicentrics in Saccharomyces cerevisiae by a mitotic recombination assay. The assay uses a diploid strain in which one homolog has a conditional centromere in addition to a wild-type centromere, and the other homolog has only the wild-type centromere; the conditional centromere is inactive when cells are grown in galactose and is activated when the cells are switched to glucose. In addition, the two homologs are distinguishable by multiple single-nucleotide polymorphisms (SNPs). Under conditions in which the conditional centromere is activated, the functionally dicentric chromosome undergoes double-stranded DNA breaks (DSBs) that can be repaired by mitotic recombination with the homolog. Such recombination events often lead to loss of heterozygosity (LOH) of SNPs that are centromere distal to the crossover. Using a PCR-based assay, we determined the position of LOH in multiple independent recombination events to a resolution of ∼4 kb. This analysis shows that dicentric chromosomes have recombination breakpoints that are broadly distributed between the two centromeres, although there is a clustering of breakpoints within 10 kb of the conditional centromere.

Phylogenetic and Molecular Epidemiological Studies Reveal Evidence of Multiple Past Recombination Events between Infectious Laryngotracheitis Viruses

In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field.

The recent recombinant evolution of a major crop pathogen, potato virus Y.

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVYNTN occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences.

An Exactly Solvable Model of Random Site-Specific Recombinations

Cre-lox and other systems are used as genetic tools to control site-specific recombination (SSR) events in genomic DNA. If multiple recombination sites are organized in a compact cluster within the same genome, a series of random recombination events may generate substantial cell specific genomic diversity. This diversity is used, for example, to distinguish neurons in the brain of the same multicellular mosaic organism, within the brainbow approach to neuronal connectome. In this paper, we study an exactly solvable statistical model for SSR operating on a cluster of recombination sites. We consider two types of recombination events: inversions and excisions. Both of these events are available in the Cre-lox system. We derive three properties of the sequences generated by multiple recombination events. First, we describe the set of sequences that can in principle be generated by multiple inversions operating on the given initial sequence. We call this description the ergodicity theorem. On the basis of this description, we calculate the number of sequences that can be generated from an initial sequence. This number of sequences is experimentally testable. Second, we demonstrate that after a large number of random inversions every sequence that can be generated is generated with equal probability. Lastly, we derive the equations for the probability to find a sequence as a function of time in the limit when excisions are much less frequent than inversions, such as in shufflon sequences.

Recombination Signals In The rpoC1 Gene Indicate Gene-Flow Between Planktothrix (Cyanoprokaryota) Species.

The delineation of species boundaries in the potentially harmful cyanobacterium Planktothrix Anagnostidis et Komárek 1988 is particularly tangled. Genetic recombination has been invoked to explain the occurrence of overlapping biological traits among recognized species. Although horizontal gene transfer is shown as a driver of diversification in this genus, clear evidence for homologous recombination at the single gene level is still lacking. Several Planktothrix strains (n=244) sampled in eight fresh water lakes in north Italy were characterized by sequencing the rpoC1 gene, a molecular marker previously proposed to discriminate between species. Six haplotypes were detected, four of which are newly described. A relevant number of rpoC1 sequences (n=54) showed evidence of homologous recombination. By comparing the sequences produced in the work presented here to those available on GenBank for the genus, multiple recombination events were tracked between haplotypes associated to P. rubescens, P. suspensa and P. agardhii, the latter being a species not found in our survey. Recombination signals were in form of (i) a vast mosaic structure present in the alignment of rpoC1 haplotypes, (ii) multiple and statistically significant paths in the split decomposition network connecting these haplotypes and (iii) many individual crossing-over events detected by means of recombination detection tests. Data suggest that the molecular evolution of the rpoC1 gene in the genus Planktothrix appears as strongly influenced by homologous recombination. In addition, rpoC1 diversity effectively tracks recombinational processes among species in the complex made up by P. rubescens, P. agardhii and P. suspensa, which are not isolated in terms of gene-flow.

A genome-wide study of recombination rate variation in Bartonella henselae

BackgroundRates of recombination vary by three orders of magnitude in bacteria but the reasons for this variation is unclear. We performed a genome-wide study of recombination rate variation among genes in the intracellular bacterium Bartonella henselae, which has among the lowest estimated ratio of recombination relative to mutation in prokaryotes.ResultsThe 1.9 Mb genomes of B. henselae strains IC11, UGA10 and Houston-1 genomes showed only minor gene content variation. Nucleotide sequence divergence levels were less than 1% and the relative rate of recombination to mutation was estimated to 1.1 for the genome overall. Four to eight segments per genome presented significantly enhanced divergences, the most pronounced of which were the virB and trw gene clusters for type IV secretion systems that play essential roles in the infection process. Consistently, multiple recombination events were identified inside these gene clusters. High recombination frequencies were also observed for a gene putatively involved in iron metabolism. A phylogenetic study of this gene in 80 strains of Bartonella quintana, B. henselae and B. grahamii indicated different population structures for each species and revealed horizontal gene transfers across Bartonella species with different host preferences.ConclusionsOur analysis has shown little novel gene acquisition in B. henselae, indicative of a closed pan-genome, but higher recombination frequencies within the population than previously estimated. We propose that the dramatically increased fixation rate for recombination events at gene clusters for type IV secretion systems is driven by selection for sequence variability.

Computational and Serologic Analysis of Novel and Known Viruses in Species Human Adenovirus D in Which Serology and Genomics Do Not Correlate

In November of 2007 a human adenovirus (HAdV) was isolated from a bronchoalveolar lavage (BAL) sample recovered from a biopsy of an AIDS patient who presented with fever, cough, tachycardia, and expiratory wheezes. To better understand the isolated virus, the genome was sequenced and analyzed using bioinformatic and phylogenomic analysis. The results suggest that this novel virus, which is provisionally named HAdV-D59, may have been created from multiple recombination events. Specifically, the penton, hexon, and fiber genes have high nucleotide identity to HAdV-D19C, HAdV-D25, and HAdV-D56, respectively. Serological results demonstrated that HAdV-D59 has a neutralization profile that is similar yet not identical to that of HAdV-D25. Furthermore, we observed a two-fold difference between the ability of HAdV-D15 and HAdV-D25 to be neutralized by reciprocal antiserum indicating that the two hexon proteins may be more similar in epitopic conformation than previously assumed. In contrast, hexon loops 1 and 2 of HAdV-D15 and HAdV-D25 share 79.13 and 92.56 percent nucleotide identity, respectively. These data suggest that serology and genomics do not always correlate.

A High Variability of Mixed Infections and Recent Recombinations of Hepatitis B Virus in Laos

In Lao PDR, where more than 8% of the population are chronic carriers of HBsAg, multiple genotypes and subgenotypes co-circulate and are prone to generate recombinant viruses. Phylogenetic analyses of multiple clones per donor revealed mixed infections of subgenotypes B1, B2, B4, C1, C5, I1 and I2 in almost 6% of HBsAg positive rejected blood donors. Recombination analyses and distance calculations furthermore showed that about 65% (17/26) of the mixed infected donors showed recombinations in the S-gene alone, involving the predominant genotypes B and C. These results suggest that, at least in Laos, hepatitis B virus (HBV) mixed infections lead to frequent recombinations. In many donors with recombinant strains, the recombinant fragment and a non-recombinant strain of the same genotype co-existed (127/185 analysed recombinant fragments). For a large proportion of these (60/127), the most closely related known virus was found, although not always exclusively, in the same donor. Recombinant virus strains are largely distinct. This is reflected in an unexpected diversity in recombination breakpoints and the relatively rare recombinations with identical recombination patterns of the same genotypes in different donors. Recent recombination events would explain the limited spread of each of the recombinants. Using a published mutation rate of 4.2×10−5 mutations per site and year, the observed minimum genetic distances of 0–0.60% between parent strain and recombinant fragment would correspond to 0–71 years of evolution from a most recent common ancestor (MRCA). Thus several lines of evidence are suggestive of recent independent recombination events, a proportion of these even occurring within the same donors. In conclusion, our analyses revealed a high variability of mixed infections as a very probable breeding ground of multiple variable recombination events in Laos that so far have not led to new dominant strains.

Novel Plasmid and Its Variant Harboring both a bla NDM-1 Gene and Type IV Secretion System in Clinical Isolates of Acinetobacter lwoffii

The spread of the bla(NDM-1) gene is gaining worldwide attentions. This gene is usually carried by large plasmids and has been discovered in diverse bacteria since it was originally found in Klebsiella pneumoniae. Here we report the complete sequences of a bla(NDM-1)-bearing plasmid, pNDM-BJ01, and its variant, pNDM-BJ02, isolated from clinical Acinetobacter lwoffii strains. The plasmid pNDM-BJ01 is 47.3 kb in size and cannot be classified into any known plasmid incompatibility group, thus representing a novel plasmid with an unknown maintenance mechanism. This plasmid contains both a bla(NDM-1) gene and a type IV secretion system (T4SS) gene cluster. The T4SS is assigned to the P-type T4SS group, which usually encode a short, rigid pilus, and the bla(NDM-1) gene is located within a composite transposon flanked by two insertion elements of ISAba125. Plasmid pNDM-BJ02 is nearly identical to pNDM-BJ01 except that one copy of the ISAba125 element is missing, and it is therefore regarded as a variant of pNDM-BJ01. Sequence alignment indicated that this bla(NDM-1)-containing composite transposon, which can also be captured by other mobile elements, was probably a product of multiple recombination events and can move as a whole by transposition.