Recapitulating Cross-Species Transmission of Simian Immunodeficiency Virus SIVcpz to Humans by Using Humanized BLT Mice.

UNLABELLED: The origins of human immunodeficiency virus type 1 (HIV-1) have been widely accepted to be the consequences of simian immunodeficiency viruses from wild chimpanzees (SIVcpz) crossing over to humans. However, there has not been any in vivo study of SIVcpz infection of humans. Also, it remains largely unknown why only specific SIVcpz strains have achieved cross-species transmission and what transmission risk might exist for those SIVcpz strains that have not been found to infect humans. Closing this knowledge gap is essential for better understanding cross-species transmission and predicting the likelihood of additional cross-species transmissions of SIV into humans. Here we show that humanized bone marrow, thymus, and liver (hu-BLT) mice are susceptible to all studied strains of SIVcpz, including the inferred ancestral viruses of pandemic and nonpandemic HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505) as well as strains that have not been found in humans (SIVcpzMT145 and SIVcpzBF1167). Importantly, the ability of SIVcpz to cross the interspecies barrier to infect humanized mice correlates with their phylogenetic distance to pandemic HIV-1. We also identified mutations of SIVcpzMB897 (Env G411R and G413R) and SIVcpzBF1167 (Env H280Q and Q380R) at 14 weeks postinoculation. Together, our results have recapitulated the events of SIVcpz cross-species transmission to humans and identified mutations that occurred during the first 16 weeks of infection, providing in vivo experimental evidence that the origins of HIV-1 are the consequence of SIVcpz crossing over to humans. This study also revealed that SIVcpz viruses whose inferred descendants have not been found in humans still have the potential to cause an HIV-1-like zoonosis. IMPORTANCE: It is believed that the origins of HIV-1 are the consequence of SIV from wild chimpanzees crossing over to humans. However, the origins of HIV-1 have been linked back to only specific SIVcpz strains. There have been no experiments that directly test the in vivo cross-species transmissibility of SIVcpz strains to humans. This is the first in vivo study of SIVcpz cross-species transmission. With the humanized-BLT mouse model, we have provided in vivo experimental evidence of multiple SIVcpz strains crossing over to humans and identified several important mutations of divergent SIVcpz strains after long-term replication in human cells. We also found that the cross-species transmission barrier of SIVcpz to humans correlates with their phylogenetic distance to pandemic HIV-1 group M. Importantly, this work provides evidence that SIVcpz viruses, whose inferred descendants have not been found in humans, still have the potential to cause a future HIV-1-like zoonotic outbreak.

Next-Generation mRNA Sequencing Reveals Pyroptosis-Induced CD4+ T Cell Death in Early Simian Immunodeficiency Virus-Infected Lymphoid Tissues.

UNLABELLED: Lymphoid tissues (LTs) are the principal sites where human immunodeficiency virus type 1 (HIV-1) replicates and virus-host interactions take place, resulting in immunopathology in the form of inflammation, immune activation, and CD4(+) T cell death. The HIV-1 pathogenesis in LTs has been extensively studied; however, our understanding of the virus-host interactions in the very early stages of infection remains incomplete. We investigated virus-host interactions in the rectal draining lymph nodes (dLNs) of rhesus macaques at different times after intrarectal inoculation (days postinoculation [dpi]) with simian immunodeficiency virus (SIV). At 3 dpi, 103 differentially expressed genes (DEGs) were detected using next-generation mRNA sequencing (RNA-seq). At 6 and 10 dpi, concomitant with increased SIV replication, 366 and 1,350 DEGs were detected, respectively, including upregulation of genes encoding proteins that play a role in innate antiviral immune responses, inflammation, and immune activation. Notably, genes (IFI16, caspase-1, and interleukin 1ß [IL-1ß]) in the canonical pyroptosis pathway were significantly upregulated in expression. We further validated increased pyroptosis using flow cytometry and found that the number of CD4(+) T cells expressing activated caspase-1 protein, the hallmark of ongoing pyroptosis, were significantly increased, which is correlated with decreased CD4(+) T cells in dLNs. Our results demonstrated that pyroptosis contributes to the CD4(+) T cell death in vivo in early SIV infection, which suggests that pyroptosis may play a pivotal role in the pathogenesis of SIV, and by extension, that of HIV-1, since pyroptosis not only induces CD4(+) T cell death but also amplifies inflammation and immune activation. Thus, blocking CD4(+) T cell pyroptosis could be a complementary treatment to antiretroviral therapy. IMPORTANCE: Although secondary lymphoid tissues (LTs) are principal sites of human immunodeficiency virus type 1 (HIV-1) replication, inflammation, immune activation, and CD4(+) T cell death, immunopathogenesis in LTs during early infection remains largely unknown. Using the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV rectal infection, we investigated early virus-host interactions. Our results revealed elevated potent host responses in early infection in LTs, including upregulation of genes involved in antiviral immune response, inflammation, and immune activation. Importantly, genes involved in the canonical pyroptosis pathway were significantly upregulated, and there was a strong correlation between CD4(+) T cell decrease and increased number of CD4(+) T cells expressing activated caspase-1 protein, demonstrating that pyroptosis contributes to CD4(+) T cell death in vivo in very early SIV infection. Our finding suggests that blocking pyroptosis may be able to decrease CD4(+) T cell loss during early SIV infection.

Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice.

Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9-11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.

A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection.

UNLABELLED: Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine. IMPORTANCE: The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

Small interfering RNA-mediated translation repression alters ribosome sensitivity to inhibition by cycloheximide in Chlamydomonas reinhardtii.

Small RNAs (sRNAs; ∼20 to 30 nucleotides in length) play important roles in gene regulation as well as in defense responses against transposons and viruses in eukaryotes. Their biogenesis and modes of action have attracted great attention in recent years. However, many aspects of sRNA function, such as the mechanism(s) of translation repression at postinitiation steps, remain poorly characterized. In the unicellular green alga Chlamydomonas reinhardtii, sRNAs derived from genome-integrated inverted repeat transgenes, perfectly complementary to the 3' untranslated region of a target transcript, can inhibit protein synthesis without or with only minimal mRNA destabilization. Here, we report that the sRNA-repressed transcripts are not altered in their polyadenylation status and they remain associated with polyribosomes, indicating inhibition at a postinitiation step of translation. Interestingly, ribosomes associated with sRNA-repressed transcripts show reduced sensitivity to translation inhibition by some antibiotics, such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in C. reinhardtii may involve alterations to the function/structural conformation of translating ribosomes. Additionally, sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, suggesting that this mechanism may have been a feature of an ancestral RNA interference machinery.

Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus.

The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors.

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic Genes.

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world's inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic "contexts", the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed "gene gangs". Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.

Effects of the synthetic corticosteroid dexamethasone on bovine herpesvirus 1 productive infection.

Sensory neurons are a primary site for life-long latency of bovine herpesvirus 1 (BoHV-1). The synthetic corticosteroid dexamethasone induces reactivation from latency and productive infection, in part because the BoHV-1 genome contains more than 100 glucocorticoid receptor (GR) responsive elements (GREs). Two GREs in the immediate early transcription unit 1 promoter are required for dexamethasone induction. Recent studies also demonstrated that the serum and glucocorticoid receptor protein kinase (SGK) family stimulated BoHV-1 replication. Consequently, we hypothesized that dexamethasone influences several aspects of productive infection. In this study, we demonstrated that dexamethasone increased expression of the immediate early protein bICP4, certain late transcripts, and UL23 (thymidine kinase) by four hours after infection. SGK1 expression and Akt phosphorylation were also stimulated during early stages of infection and dexamethasone treatment further increased this effect. These studies suggest that stress, as mimicked by dexamethasone treatment, has the potential to stimulate productive infection by multiple pathways.

Distinct transcriptome profiles of Gag-specific CD8+ T cells temporally correlated with the protection elicited by SIVΔnef live attenuated vaccine.

The live attenuated vaccine (LAV) SIVmac239Δnef (SIVΔnef) confers the best protection among all the vaccine modalities tested in rhesus macaque model of HIV-1 infection. This vaccine has a unique feature of time-dependent protection: macaques are not protected at 3-5 weeks post vaccination (WPV), whereas immune protection emerges between 15 and 20 WPV. Although the exact mechanisms of the time-dependent protection remain incompletely understood, studies suggested that both cellular and humoral immunities contribute to this time-dependent protection. To further elucidate the mechanisms of protection induced by SIVΔnef, we longitudinally compared the global gene expression profiles of SIV Gag-CM9+ CD8+ (Gag-specific CD8+) T cells from peripheral blood of Mamu-A*01+ rhesus macaques at 3 and 20 WPV using rhesus microarray. We found that gene expression profiles of Gag-specific CD8+ T cells at 20 WPV are qualitatively different from those at 3 WPV. At 20 WPV, the most significant transcriptional changes of Gag-specific CD8+ T cells were genes involved in TCR signaling, differentiation and maturation toward central memory cells, with increased expression of CCR7, TCRα, TCRß, CD28 and decreased expression of CTLA-4, IFN-γ, RANTES, granzyme A and B. Our study suggests that a higher quality of SIV-specific CD8+ T cells elicited by SIVΔnef over time contributes to the maturation of time-dependent protection.

Characterization of founder viruses in very early SIV rectal transmission.

A better understanding of HIV-1 transmission is critical for developing preventative strategies. To that end, we analyzed 524 full-length env sequences of SIVmac251 at 6 and 10 days post intrarectal infection of rhesus macaques. There was no tissue compartmentalization of founder viruses across plasma, rectal and distal lymphatic tissues for most animals; however one animal has evidence of virus tissue compartmentalization. Despite identical viral inoculums, founder viruses were animal-specific, primarily derived from rare variants in the inoculum, and have a founder virus signature that can distinguish dominant founder variants from minor founder or untransmitted variants in the inoculum. Importantly, the sequences of post-transmission defective viruses were phylogenetically associated with competent viral variants in the inoculum and were mainly converted from competent viral variants by frameshift rather than APOBEC mediated mutations, suggesting the converting the transmitted viruses into defective viruses through frameshift mutation is an important component of rectal transmission bottleneck. SIGNIFICANCE: Anorectal receptive intercourse is a common route of HIV-1 transmission and a better understanding of the transmission mechanisms is critical for developing HIV-1 preventative strategies. Here, we report that there is no tissue compartmentalization of founder viruses during very early rectal transmission of SIV in the majority of rhesus macaques and founder viruses are preferentially derived from rare variant in the inoculum. We also found that founder viruses are animal-specific despite identical viral inoculums. After viruses cross the mucosal barriers, the host further reduces viral diversity by converting some of the transmitted functional viruses into defective viruses through frameshift rather than APOBEC derived mutations. To our knowledge, this is the first study of founder viruses at multiple tissue sites during very early rectal transmission.

Coccolithoviruses: A Review of Cross-Kingdom Genomic Thievery and Metabolic Thuggery.

Coccolithoviruses (Phycodnaviridae) infect and lyse the most ubiquitous and successful coccolithophorid in modern oceans, Emiliania huxleyi. So far, the genomes of 13 of these giant lytic viruses (i.e., Emiliania huxleyi viruses-EhVs) have been sequenced, assembled, and annotated. Here, we performed an in-depth comparison of their genomes to try and contextualize the ecological and evolutionary traits of these viruses. The genomes of these EhVs have from 444 to 548 coding sequences (CDSs). Presence/absence analysis of CDSs identified putative genes with particular ecological significance, namely sialidase, phosphate permease, and sphingolipid biosynthesis. The viruses clustered into distinct clades, based on their DNA polymerase gene as well as full genome comparisons. We discuss the use of such clustering and suggest that a gene-by-gene investigation approach may be more useful when the goal is to reveal differences related to functionally important genes. A multi domain "Best BLAST hit" analysis revealed that 84% of the EhV genes have closer similarities to the domain Eukarya. However, 16% of the EhV CDSs were very similar to bacterial genes, contributing to the idea that a significant portion of the gene flow in the planktonic world inter-crosses the domains of life.

Characterization of a new chlorovirus type with permissive and non-permissive features on phylogenetically related algal strains.

A previous report indicated that prototype chlorovirus PBCV-1 replicated in two Chlorella variabilis algal strains, NC64A and Syngen 2-3, that are ex-endosymbionts isolated from the protozoan Paramecium bursaria. Surprisingly, plaque-forming viruses on Syngen 2-3 lawns were often higher than on NC64A lawns from indigenous water samples. These differences led to the discovery of viruses that exclusively replicate in Syngen 2-3 cells, named Only Syngen (OSy) viruses. OSy-NE5, the prototype virus for the proposed new species, had a linear dsDNA genome of 327kb with 44-nucleotide-long, incompletely base-paired, covalently closed hairpin ends. Each hairpin structure was followed by an identical 2612 base-paired inverted sequence after which the DNA sequence diverged. OSy-NE5 encoded 357 predicted CDSs and 13 tRNAs. Interestingly, OSy-NE5 attached to and initiated infection in NC64A cells but infectious progeny viruses were not produced; thus OSy-NE5 replication in NC64A is blocked at some later stage of replication.

Genome-Wide Search for Host Association Factors during Ovine Progressive Pneumonia Virus Infection.

Ovine progressive pneumonia virus (OPPV) is an important virus that causes serious diseases in sheep and goats with a prevalence of 36% in the USA. Although OPPV was discovered more than half of a century ago, little is known about the infection and pathogenesis of this virus. In this report, we used RNA-seq technology to conduct a genome-wide probe for cellular factors that are associated with OPPV infection. A total of approximately 22,000 goat host genes were detected of which 657 were found to have been significantly up-regulated and 889 down-regulated at 12 hours post-infection. In addition to previously known restriction factors from other viral infections, a number of factors which may be specific for OPPV infection were uncovered. The data from this RNA-seq study will be helpful in our understanding of OPPV infection, and also for further study in the prevention and intervention of this viral disease.

Virus-host mucosal interactions during early SIV rectal transmission.

To deepen our understanding of early rectal transmission of HIV-1, we studied virus-host interactions in the rectal mucosa using simian immunodeficiency virus (SIV)-Indian rhesus macaque model and mRNA deep sequencing. We found that rectal mucosa actively responded to SIV as early as 3 days post-rectal inoculation (dpi) and mobilized more robust responses at 6 and 10 dpi. Our results suggest that the failure of the host to contain virus replication at the portal of entry is attributable to both a high-level expression of lymphocyte chemoattractant, proinflammatory and immune activation genes, which can recruit and activate viral susceptible target cells into mucosa; and a high-level expression of SIV accessory genes, which are known to be able to counter and evade host restriction factors and innate immune responses. This study provides new insights into the mechanism of rectal transmission.

Host genetics and diet, but not immunoglobulin A expression, converge to shape compositional features of the gut microbiome in an advanced intercross population of mice.

BACKGROUND: Individuality in the species composition of the vertebrate gut microbiota is driven by a combination of host and environmental factors that have largely been studied independently. We studied the convergence of these factors in a G10 mouse population generated from a cross between two strains to search for quantitative trait loci (QTLs) that affect gut microbiota composition or ileal Immunoglobulin A (IgA) expression in mice fed normal or high-fat diets. RESULTS: We found 42 microbiota-specific QTLs in 27 different genomic regions that affect the relative abundances of 39 taxa, including four QTL that were shared between this G10 population and the population previously studied at G4. Several of the G10 QTLs show apparent pleiotropy. Eight of these QTLs, including four at the same site on chromosome 9, show significant interaction with diet, implying that diet can modify the effects of some host loci on gut microbiome composition. Utilization patterns of IghV variable regions among IgA-specific mRNAs from ileal tissue are affected by 54 significant QTLs, most of which map to a segment of chromosome 12 spanning the Igh locus. Despite the effect of genetic variation on IghV utilization, we are unable to detect overlapping microbiota and IgA QTLs and there is no significant correlation between IgA variable pattern utilization and the abundance of any of the taxa from the fecal microbiota. CONCLUSIONS: We conclude that host genetics and diet can converge to shape the gut microbiota, but host genetic effects are not manifested through differences in IgA production

Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats.

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of ß-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and ß-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress.

Multiple genome alignment based on longest path in directed acyclic graphs.

In this paper, we present a simple and efficient algorithm for multiple genome sequence alignment. Sequences of Maximal Unique Matches (MUMs) are first transformed into a multi-bipartite diagram. The diagram is then converted into a Directed Acyclic Graph (DAG). Therefore, finding the alignment is reduced to finding the longest path in the DAG, which is solvable in linear time. The experiments show that the algorithm can correctly find the alignment, and runs faster than MGA and EMAGEN. In addition, our algorithm can handle the alignments with overlapping MUMs and has both weighted and unweighted options. It provides the flexibility for the alignments depending on different needs.