Recombination Breakpoints Research Articles

Ancestral male recombination in Drosophila albomicans produced geographically restricted neo-Y chromosome haplotypes varying in age and onset of decay.

Male Drosophila typically have achiasmatic meiosis, and fusions between autosomes and the Y chromosome have repeatedly created non-recombining neo-Y chromosomes that degenerate. Intriguingly, Drosophila nasuta males recombine, but their close relative D. albomicans reverted back to achiasmy after evolving neo-sex chromosomes. Here we use genome-wide polymorphism data to reconstruct the complex evolutionary history of neo-sex chromosomes in D. albomicans and examine the effect of recombination and its cessation on the initiation of neo-Y decay. Population and phylogenomic analyses reveal three distinct neo-Y types that are geographically restricted. Due to ancestral recombination with the neo-X, overall nucleotide diversity on the neo-Y is similar to the neo-X but severely reduced within neo-Y types. Consistently, the neo-Y chromosomes fail to form a monophyletic clade in sliding window trees outside of the region proximal to the fusion. Based on tree topology changes, we inferred the recombination breakpoints that produced haplotypes specific to each neo-Y type. We show that recombination became suppressed at different time points for the different neo-Y haplotypes. Haplotype age correlates with onset of neo-Y decay, and older neo-Y haplotypes show more fixed gene disruption via frameshift indels and down-regulation of neo-Y alleles. Genes are downregulated independently on the different neo-Ys, but are depleted of testes-expressed genes across all haplotypes. This indicates that genes important for male function are initially shielded from degeneration. Our results offer a time course of the early progression of Y chromosome evolution, showing how the suppression of recombination, through the reversal to achiasmy in D. albomicans males, initiates the process of degeneration.

Identification of a conserved ph1b-mediated 5DS–5BS crossing over site in soft-kernel durum wheat (Triticum turgidum subsp. durum) lines

Genetic recombination is the major mechanism and basis of genetic diversity and crop improvement. Recombination typically occurs between homologous chromosomes. In wheat, however, it can also occur between homoeologous chromosomes if a functional Ph1 (Pairing homoeologous-1) locus is absent (ph1b). Recently, the genes for soft kernel trait on the distal 5DS chromosome of common wheat were transferred into chromosome 5BS of durum wheat through ph1b-mediated recombination. Several independent 5DS–5BS recombination lines carrying the 5DS translocation were obtained. However, the specific size of the translocation and region where the translocation breakpoint occurred was not determined for any of the lines. In the present study, four independent 5DS–5BS recombination lines were investigated and the translocation breakpoints were fine-mapped and sequenced. All the analyzed lines lost a 5BS fragment of ~ 20,742,425 bp and gained a 5DS fragment of ~ 28,163,252 bp. The recombination breakpoint in each line was located within a predicted gene in a conserved 39 bp region, suggesting the presence of a recombination hotspot. The information obtained was then used to develop a KASP marker diagnostic for the 5DS–5BS translocation. Finally, a subset of the soft durum wheat lines exhibiting varying degrees of kernel hardness was analyzed through genomic in situ hybridization (GISH) and scanning electron microscopy (SEM). From both the GISH and SEM analyses no major differences were detected among the lines, indicating that factors other than ph1b-mediated recombination or endosperm morphology were responsible for the variation in kernel hardness.

Identification of a Novel HIV-1 Second-Generation (CRF01_AE/B) Among Men Who Have Sex with Men in Tianjin, China.

In this study, we report a novel HIV-1 second-generation recombinant form composed of CRF01_AE and subtype B detected from a married HIV-positive male subject infected through homosexual behavior in Tianjin in northern China. The near full-length genome analyses showed that two regions of subtype B inserted into the CRF01_AE backbone with four recombinant breakpoints observed in the pol gene region. Subregion tree analyses demonstrated that the CRF01_AE regions of the recombinant were greatly clustered with the CRF01_AE subcluster 4 lineage, which was found primarily among men who have sex with men (MSM) in northern China. To the best of our knowledge, this is the first detection of a novel HIV-1 second-generation recombinant form (CRF01AE/B) in Tianjin, which indicates active transmission networks of HIV-1 infection among MSM in this region. The emergence of the novel second-generation recombinant form highlights the increasing complexity of HIV-1 epidemic among MSM population and the importance to monitor potential novel circulating recombinant forms.

Frog Virus 3 Genomes Reveal Prevalent Recombination between Ranavirus Lineages and Their Origins in Canada.

Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTVs have been reported only in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within open reading frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (<100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread.IMPORTANCE Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade.

Hepatitis E virus in Tibetan pigs on the Qinghai-Tibetan Plateau: detection, molecular characterization and infection of BALB/c mice.

This study was performed to investigate the prevalence and genetic variation of hepatitis E virus (HEV) in Tibetan pigs and to determine its ability to infect mice. A total of 38 out of 229 (16.59%; 95% CI = 12.00%-22.10%) fecal samples from Tibetan pigs from the Qinghai-Tibetan Plateau in 2018 were positive for HEV RNA, which was detected by RT-nPCR. Significantly different detection rates were observed between samples from diarrheic and clinically healthy animals (OR = 9.56; 95% CI, 2.84-32.14; p < 0.001), suggesting a potential association between HEV infection and diarrhea in Tibetan pigs. Phylogenetic analysis showed that the HEV isolates were clustered into subtypes 4a (31 samples), 4b (1), 4d (2), and 4j (4). HEV-4a was the predominant subtype, indicating that it might be circulating in Tibetan pigs. Nine complete HEV genome sequences obtained from Tibetan pigs were found by phylogenetic analysis to be closely related to those of genotype 4 HEV isolates from humans. Two recombinant events were identified in both HEV-4a strains; a novel recombination breakpoint was first identified at the 3' end of the ORF2 region in the SWU/L9/2018 strain, and a common recombination region was found at the junction of the ORF1 and ORF2 regions in the SWU/31-12/2018 strain. Furthermore, HEV-4a could be detected in all BALB/c mice that were experimentally infected by gavage and contact exposure. The information presented here about the prevalence and genotype diversity of HEV from Tibetan pigs provides important insights into the epidemic features of HEV on the Qinghai-Tibetan Plateau.

Genotyping by low-coverage whole-genome sequencing in intercross pedigrees from outbred founders: a cost-efficient approach

BackgroundExperimental intercrosses between outbred founder populations are powerful resources for mapping loci that contribute to complex traits i.e. quantitative trait loci (QTL). Here, we present an approach and its accompanying software for high-resolution reconstruction of founder mosaic genotypes in the intercross offspring from such populations using whole-genome high-coverage sequence data on founder individuals (~ 30×) and very low-coverage sequence data on intercross individuals (< 0.5×). Sets of founder-line informative markers were selected for each full-sib family and used to infer the founder mosaic genotypes of the intercross individuals. The application of this approach and the quality of the estimated genome-wide genotypes are illustrated in a large F2 pedigree between two divergently selected lines of chickens.ResultsWe describe how we obtained whole-genome genotype data for hundreds of individuals in a cost- and time-efficient manner by using a Tn5-based library preparation protocol and an imputation algorithm that was optimized for this application. In total, 7.6 million markers segregated in this pedigree and, within each full-sib family, between 10.0 and 13.7% of these were fully informative, i.e. fixed for alternative alleles in the founders from the divergent lines, and were used for reconstruction of the offspring mosaic genotypes. The genotypes that were estimated based on the low-coverage sequence data were highly consistent (> 95% agreement) with those obtained using individual single nucleotide polymorphism (SNP) genotyping. The estimated resolution of the inferred recombination breakpoints was relatively high, with 50% of them being defined on regions shorter than 10 kb.ConclusionsA method and software for inferring founder mosaic genotypes in intercross offspring from low-coverage whole-genome sequencing in pedigrees from heterozygous founders are described. They provide high-quality, high-resolution genotypes in a time- and cost-efficient manner. The software is freely available at https://github.com/CarlborgGenomics/Stripes.

A54 Genomic analysis of camel-HKU23 in Nigeria dromedary camels reveals strain-specific cross-species recombination

Coronaviruses (CoVs) are enveloped, single stranded, positive-sense RNA viruses with a large genomic size of 26–32 kilobases. The first human CoV identified in the 1960s was isolated from patients presenting with common cold symptoms. Subsequent epidemic outbreaks of novel zoonotic CoV transmission were reported, examples including HCoV-229E (229E), HCoV-OC43 (OC43), severe acute respiratory syndrome, and Middle East respiratory syndrome (MERS). The ongoing outbreak of MERS in the Middle East is originating from a zoonotic source of dromedary camels. Surveillance later revealed that three CoV species—HCoV-229E (229E), camel-HKU23, and MERS-CoV—were co-circulating in Saudi Arabia dromedary camels. Camel-HKU23 belongs to Group 2a CoV, which also includes human coronavirus OC43, bovine coronavirus, and porcine hemagglutinating encephalomyelitis virus. Recombination, resulting in the generation of different novel genotypes, has been reported previously among these CoVs. Our surveillance of dromedary camels slaughtered in a major abattoir in Nigeria identified camel-HKU23 from nasal swab samples with a prevalence of 2.2 per cent. Phylogenetic analysis showed Nigeria camel-HKU23 is distinct from those previously identified in Saudi Arabia, while still genetically similar, as they share a monophyletic origin. Recombination analysis of Nigeria camel-HKU23 revealed two recombination breakpoints at positions of 22774–24100 base pairs (bp) and 28224–29362 bp. Recombination breakpoint at position 22774, encoding the Group 2a CoV-specific hemagglutinin esterase gene, exhibited high bootstrap support for clustering with RbCoV HKU14, which was previously detected in domestic rabbits in China. The recombination signal is only observed in Nigeria camel-HKU23, suggesting a regional varied evolutionary history of camel-HKU23. Our findings extended the knowledge of the evolutionary relationship among Group 2a CoVs. Further surveillance in other African camels will be important to elucidate the evolution of camel-HKU23.

Identification of hepatitis B virus genotype A/E recombinants in Ghana.

Hepatitis B virus (HBV) exhibits a high degree of heterogeneity with at least 10 genotypes (A-J) identified to date. Intergenotypic recombination is relatively common. Previously, we investigated HBV drug resistance in HIV/HBV co-infected individuals in Ghana. After identifying multiple circulating genotypes and a novel D/E recombinant, we sought to determine if additional individuals were also infected with recombinant HBV. Partial genome sequences from three individuals were initially identified as genotype A4. Full-length HBV genomes were obtained using rolling circle amplification followed by PCR and shown to cluster with known A/E recombinant viruses. Similar recombination breakpoints were observed in these three individuals suggesting local spread of this novel recombinant HBV in Ghana.

Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant Form (CRF01_AE/B) from Men Who Have Sex with Men in Guangxi Zhuang Autonomous Region, China.

We report here a novel HIV-1 recombinant form (18GXD4705) composed of CRF01_AE and subtype B, acquired from an unmarried HIV-positive young man subject infected through homosexual contact in Guangxi Province of eastern China. The phylogenetic analysis of the near full-length genome of 18GXD4705 indicated that one subtype B segment was inserted into the CRF01_AE backbone, with one recombinant breakpoint demonstrated in the pol region. The CRF01_AE region (I and III) of recombinant correlated with a previously reported subcluster 4 lineage. The B subregions (II) are greatly clustered together, with B strain references. The continued generation of this novel recombinant increases the genetic complexity and diversity of the HIV epidemic in Guangxi. In addition, further molecular epidemiological investigations should be conducted to continuously monitor the dynamic transmission of HIV-1 in the region.

Genomic Characterization of a Novel HIV-1 CRF01_AE/07_BC Recombinant Virus from a Married Man Who Has Sex with Men in Tianjin, China.

We report in this study a novel HIV-1 second-generation recombinant form (TJIH0172) composed of CRF01_AE and CRF07_BC isolated from a married HIV-positive male subject infected through homosexual behavior in Tianjin, China. The phylogenetic analysis of the near full-length genome of TJIH0172 reveals that one region of CRF07_BC inserted into the CRF01_AE backbone with two recombinant breakpoints observed in the vpu and env gene regions, respectively. The CRF01_AE regions (the regions I and III) of the recombinant are greatly clustered with the CRF01_AE subcluster 4 lineage, which is mainly circulating among men who have sex with men (MSM) in northern China. The CRF07_BC region (II) is clustered with two sequences (JX960600 and KF250366), which were discovered in the MSM population in Liaoning Province and Beijing city in northern China, respectively. The emergence of the novel recombinant strain from a married man who has sex with men in Tianjin, China, highlights the increasing complexity of the HIV-1 epidemic between MSM and their female partners and further molecular epidemiological investigation should be taken to track married MSM and their female partners to prevent HIV transmit from HIV high-risk populations to general populations.

Hepatitis C virus genotype 1 and 2 recombinant genomes and the phylogeographic history of the 2k/1b lineage.

Recombination is an important driver of genetic diversity, though it is relatively uncommon in hepatitis C virus (HCV). Recent investigation of sequence data acquired from HCV clinical trials produced twenty-one full-genome recombinant viruses belonging to three putative inter-subtype forms 2b/1a, 2b/1b, and 2k/1b. The 2k/1b chimera is the only known HCV circulating recombinant form (CRF), provoking interest in its genetic structure and origin. Discovered in Russia in 1999, 2k/1b cases have since been detected throughout the former Soviet Union, Western Europe, and North America. Although 2k/1b prevalence is highest in the Caucasus mountain region (i.e., Armenia, Azerbaijan, and Georgia), the origin and migration patterns of CRF 2k/1b have remained obscure due to a paucity of available sequences. We assembled an alignment which spans the entire coding region of the HCV genome containing all available 2k/1b sequences (>500 nucleotides; n = 109) sampled in ninteen countries from public databases (102 individuals), additional newly sequenced genomic regions (from 48 of these 102 individuals), unpublished isolates with newly sequenced regions (5 additional individuals), and novel complete genomes (2 additional individuals) generated in this study. Analysis of this expanded dataset reconfirmed the monophyletic origin of 2k/1b with a recombination breakpoint at position 3,187 (95% confidence interval: 3,172–3,202; HCV GT1a reference strain H77). Phylogeography is a valuable tool used to reveal viral migration dynamics. Inference of the timed history of spread in a Bayesian framework identified Russia as the ancestral source of the CRF 2k/1b clade. Further, we found evidence for migration routes leading out of Russia to other former Soviet Republics or countries under the Soviet sphere of influence. These findings suggest an interplay between geopolitics and the historical spread of CRF 2k/1b.

G-quadruplexes may determine the landscape of recombination in HSV-1

BackgroundSeveral lines of evidence suggest that recombination plays a central role in replication and evolution of herpes simplex virus-1 (HSV-1). G-quadruplex (G4)-motifs have been linked to recombination events in human and microbial genomes, but their role in recombination has not been studied in DNA viruses.ResultsThe availability of near full-length sequences from 40 HSV-1 recombinant strains with exact position of the recombination breakpoints provided us with a unique opportunity to investigate the role of G4-motifs in recombination among herpes viruses. We mapped the G4-motifs in the parental and all the 40 recombinant strains. Interestingly, the genome-wide distribution of breakpoints closely mirrors the G4 densities in the HSV-1 genome; regions of the genome with higher G4 densities had higher number of recombination breakpoints. Biophysical characterization of oligonucleotides from a subset of predicted G4-motifs confirmed the formation of G-quadruplex structures. Our analysis also reveals that G4-motifs are enriched in regions flanking the recombination breakpoints. Interestingly, about 11% of breakpoints lie within a G4-motif, making these DNA secondary structures hotspots for recombination in the HSV-1 genome. Breakpoints within G4-motifs predominantly lie within G4-clusters rather than individual G4-motifs. Of note, we identified the terminal guanosine of G4-clusters at the boundaries of the UL (unique long) region on either side of the OriL (origin of replication within UL) represented the commonest breakpoint among the HSV-1 recombinants.ConclusionOur findings suggest a correlation between the HSV-1 recombination landscape and the distribution of G4-motifs and G4-clusters, with possible implications for the evolution of DNA viruses.

Characterization of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Virus Among Men Who Have Sex with Men in Tianjin, China.

We report here a novel human immunodeficiency virus type 1 (HIV-1) second-generation recombinant form comprising CRF01_AE and CRF07_BC isolated from an HIV-positive male subject infected among men who have sex with men (MSM) in Tianjin in north China. The phylogenetic analysis of the near full-length genome of TJIH0345 reveals that three regions of CRF01_AE were inserted into the CRF07_BC backbone with five recombinant breakpoints observed in the pol, vif, vpu, and env gene regions whose genome structure is distinctly different from other circulating recombinant forms and unique recombinant forms previously reported. The continued emergence of the novel recombinant strain in Tianjin, China, highlights the increasing complexity of the HIV-1 epidemic among the MSM population, and further molecular epidemiological investigation should be done to track the genetic evolution of HIV-1 strains to prevent HIV transmission.

Near-Full-Length Genetic Characterization of a Novel HIV-1 Unique Recombinant with Similarities to A1, CRF01_AE, and CRFO2_AG Viruses in Yaoundé, Cameroon.

Variations in the HIV genome influence HIV/AIDS epidemiology. We report here a novel HIV-1 unique recombinant form (URF) isolated from an HIV-infected female (NACMR092) in Cameroon, based on the analyses of near-full-length viral genome (partial gag, full-length pol, env, tat, rev, vif, vpr, vpu, and nef genes, and partial 3'-long terminal repeat). Phylogeny, recombination breakpoints, and recombination map analyses showed that NACMR092 was infected with a mosaic URF that had eight breakpoints (two in gag, one in pol, one in vpr, two in env, and two in the nef regions), nine subgenomic regions, and included fragments that had important similarities with HIV-1 subtypes A1, CRF02_AG, and CRF01_AE. This novel mosaic URF underscores complex recombination events occurring between HIV-1 subtypes circulating in Cameroon. Continued monitoring and detection of such recombinants and accurate classification of HIV genotype is important for tracking viral molecular epidemiology and antigenic diversity.

Zucchini yellow mosaic virus Genomic Sequences from Papua New Guinea: Lack of Genetic Connectivity with Northern Australian or East Timorese Genomes, and New Recombination Findings.

Zucchini yellow mosaic virus (ZYMV) isolates were obtained in Papua New Guinea (PNG) from cucumber (Cucumis sativus) or pumpkin (Cucurbita spp.) plants showing mosaic symptoms growing at Kongop in the Mount Hagen District, Western Highlands Province, or Zage in the Goroka District, Eastern Highlands Province. The samples were blotted onto FTA cards, which were sent to Australia, where they were subjected to high-throughput sequencing. When the coding regions of the nine new ZYMV genomic sequences found were compared with those of 64 other ZYMV sequences from elsewhere, they grouped together, forming new minor phylogroup VII within ZYMV's major phylogroup A. Genetic connectivity was lacking between ZYMV genomic sequences from PNG and its neighboring countries, Australia and East Timor; the closest match between a PNG and any other genomic sequence was a 92.8% nucleotide identity with a sequence in major phylogroup A's minor phylogroup VI from Japan. When the RDP5.2 recombination analysis program was used to compare 66 ZYMV sequences, evidence was obtained of 30 firm recombination events involving 41 sequences, and all isolates from PNG were recombinants. There were 21 sequences without recombination events in major phylogroup A, whereas there were only 4 such sequences within major phylogroup B. ZYMV's P1, Cl, N1a-Pro, P3, CP, and NIb regions contained the highest evidence of recombination breakpoints. Following removal of recombinant sequences, seven minor phylogroups were absent (I, III, IV, V, VI, VII, and VIII), leaving only minor phylogroups II and IX. By contrast, when a phylogenetic tree was constructed using recombinant sequences with their recombinationally derived tracts removed before analysis, five previous minor phylogroups remained unchanged within major phylogroup A (II, III, IV, V, and VII) while four formed two new merged phylogroups (I/VI and VIII/IX). Absence of genetic connectivity between PNG, Australian, and East Timorese ZYMV sequences, and the 92.8% nucleotide identity between a PNG sequence and the closest sequence from elsewhere, suggest that a single introduction may have occurred followed by subsequent evolution to adapt to the PNG environment. The need for enhanced biosecurity measures to protect against potentially damaging virus movements crossing the seas separating neighboring countries in this region of the world is discussed.

Novel subgenotype D11 of hepatitis B virus in NaPo County, Guangxi, bordering Vietnam.

Hepatitis B virus has been classified into 10 genotypes and 48 subgenotypes worldwide. We found previously, through polymerase chain reaction (PCR) amplification of a sample collected in 2011, that an HBsAg carrier was infected with two genotypes (B and D) of HBV. We carried out cloning, sequencing and phylogenetic analysis of the complete genomes and, for confirmation, analysed a sample collected from the same individual in 2018. Fifteen complete sequences were obtained from each sample. The carrier was infected in 2011 by genotypes B and D and by various recombinants, but only genotype D was present in 2018. The major and minor parents of the recombinants are genotypes B and D, respectively, although the recombination breakpoints vary among them. All 23 genotype D isolates form a cluster, branching out from other subgenotype D sequences and supported by a 100 % bootstrap value. Based on complete genome sequences, almost all of the estimated intragroup nucleotide divergence values between our isolates and HBV subgenotypes D1-D10 exceed 4 %. Compared to the other subgenotypes (D1-D10), 35 unique amino acids were present in our isolates. Our data provide evidence for a novel subgenotype, provisionally designated HBV subgenotype D11.

Complete genome characterization and population dynamics of potato virus Y-NTN strain from India.

Potato virus Y (PVY) is a major threat to potato cultivation worldwide. PVY exists as biologically and genetically distinct strains and causes varying degrees of pathogenicity and a wide range of symptoms in potato. Knowledge of the nature of PVY strains is essential for breeding PVY resistant cultivars that are durable against a wide range of strains. We report the complete genome of a PVY potato isolate (JK12) characterised from the potato production areas of Jammu and Kashmir, India. Nucleotide sequence comparisons and phylogenetic analysis with known PVY strains revealed that the isolate belongs to the NTN strain of PVY. At the whole genome sequence level, the JK12 isolate shared the highest identity (99.42%) with PVY-NTN strains reported from Germany, followed by those from United Kingdom (99.34%) and Japan (99.33%). Recombination detection analysis identified two recombination break points and JK12 appeared to have originated from a recombination event between a PVY-N strain from Belgium as a major parent and a PVY-O strain from China as the minor parent. Our results suggest possible mutation and recombination could be the basis for the evolution and the subsequent establishment of NTN in this region. Furthermore, a global evolutionary lineage analysis of all the known PVY strains showed relatively low nucleotide diversity among the PVY-NTN strains. Neutrality tests showed that all the genotypes of PVY are undergoing purifying selection suggesting population expansion of PVY. This is the first report of complete genomic characterization of an NTN strain of PVY isolated from commercial potato fields in India. The implications of the emergence of this strain in the Indian context are discussed.

Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function.

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.

Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants.

Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

Genomic characterization of a novel recombinant porcine astrovirus isolated in northeastern China.

Porcine astroviruses (PAstVs), are widely distributed viruses that are highly prevalent in swine herds. In this study, a novel type 4 porcine astrovirus strain (designated as PAstV4/Tianjin/2018) was identified in a fecal sample from a diarrheal piglet in Tianjin, China and its complete genomic sequence was determined by RT-PCR. Sequence analysis showed that this strain had a capsid protein with a highly variable C-terminal domain, a typical ribosomal frameshifting signal, and a conserved subgenomic promoter sequence. Recombination analysis indicated that PAstV4/Tianjin/2018 was a novel recombinant strain, and a recombination breakpoint was identified at nt position 4220 of the genome. The novel recombinant porcine astrovirus identified in China will be useful for understanding the origin, genetic diversity, and evolution of enteric viruses.