jpHMM is a very accurate and widely used tool for recombination detection in genomic sequences of HIV-1. Here, we present an extension of jpHMM to analyze recombinations in viruses with circular genomes such as the hepatitis B virus (HBV). Sequence analysis of circular genomes is usually performed on linearized sequences using linear models. Since linear models are unable to model dependencies between nucleotides at the 5′- and 3′-end of a sequence, this can result in inaccurate predictions of recombination breakpoints and thus in incorrect classification of viruses with circular genomes. The proposed circular jpHMM takes into account the circularity of the genome and is not biased against recombination breakpoints close to the 5′- or 3′-end of the linearized version of the circular genome. It can be applied automatically to any query sequence without assuming a specific origin for the sequence coordinates. We apply the method to genomic sequences of HBV and visualize its output in a circular form. jpHMM is available online at http://jphmm.gobics.de for download and as a web server for HIV-1 and HBV sequences.

ith great interest we read the manuscript entitled “Isolation,Identification, and Characterization of Novel Arenaviruses,the Etiological Agent of Boid Inclusion Body Disease” by Hetzeland colleagues (1). This very well performed and comprehensivestudy about the role of divergent arenaviruses in inclusion bodydisease (IBD) of boid snakes clearly shows a causal relationshipbetween arenaviruses (Avs) and IBD

Zoonotic infectious diseases such as influenza continue to pose a grave threat to human health. However, the factors that mediate the emergence of RNA viruses such as influenza A virus (IAV) are still incompletely understood. Phylogenetic inference is crucial to reconstructing the origins and tracing the flow of IAV within and between hosts. Here we show that explicitly allowing IAV host lineages to have independent rates of molecular evolution is necessary for reliable phylogenetic inference of IAV and that methods that do not do so, including ‘relaxed’ molecular clock models, can be positively misleading. A phylogenomic analysis using a host-specific local clock model recovers extremely consistent evolutionary histories across all genomic segments and demonstrates that the equine H7N7 lineage is a sister clade to strains from birds—as well as those from humans, swine and the equine H3N8 lineage—sharing an ancestor with them in the mid to late 1800s. Moreover, major western and eastern hemisphere avian influenza lineages inferred for each gene coalesce in the late 1800s. On the basis of these phylogenies and the synchrony of these key nodes, we infer that the internal genes of avian influenza virus (AIV) underwent a global selective sweep beginning in the late 1800s, a process that continued throughout the twentieth century and up to the present. The resulting western hemispheric AIV lineage subsequently contributed most of the genomic segments to the 1918 pandemic virus and, independently, the 1963 equine H3N8 panzootic lineage. This approach provides a clear resolution of evolutionary patterns and processes in IAV, including the flow of viral genes and genomes within and between host lineages.

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated in Uganda in 1947. Although entomological and virologic surveillance have reported ZIKV enzootic activity in diverse countries of Africa and Asia, few human cases were reported until 2007, when a Zika fever epidemic took place in Micronesia. In the context of West Africa, the WHO Collaborating Centre for Arboviruses and Hemorrhagic Fever at Institut Pasteur of Dakar (http://www.pasteur.fr/recherche/banques/CRORA/) reports the periodic circulation of ZIKV since 1968. Despite several reports on ZIKV, the genetic relationships among viral strains from West Africa remain poorly understood. To evaluate the viral spread and its molecular epidemiology, we investigated 37 ZIKV isolates collected from 1968 to 2002 in six localities in Senegal and Côte d'Ivoire. In addition, we included strains from six other countries. Our results suggested that these two countries in West Africa experienced at least two independent introductions of ZIKV during the 20th century, and that apparently these viral lineages were not restricted by mosquito vector species. Moreover, we present evidence that ZIKV has possibly undergone recombination in nature and that a loss of the N154 glycosylation site in the envelope protein was a possible adaptive response to the Aedes dalzieli vector.

With the emergence of leishmaniasis in new regions around the world, molecular epidemiological methods with adequate discriminatory power, reproducibility, high throughput and inter-laboratory comparability are needed for outbreak investigation of this complex parasitic disease. As multilocus sequence analysis (MLSA) has been projected as the future gold standard technique for Leishmania species characterization, we propose a MLSA panel of six housekeeping gene loci (6pgd, mpi, icd, hsp70, mdhmt, mdhnc) for investigating intraspecific genetic variation of L. (Viannia) braziliensis strains and compare the resulting genetic clusters with several epidemiological factors relevant to outbreak investigation. The recent outbreak of cutaneous leishmaniasis caused by L. (V.) braziliensis in the southern Brazilian state of Santa Catarina is used to demonstrate the applicability of this technique. Sequenced fragments from six genetic markers from 86 L. (V.) braziliensis strains from twelve Brazilian states, including 33 strains from Santa Catarina, were used to determine clonal complexes, genetic structure, and phylogenic networks. Associations between genetic clusters and networks with epidemiological characteristics of patients were investigated. MLSA revealed epidemiological patterns among L. (V.) braziliensis strains, even identifying strains from imported cases among the Santa Catarina strains that presented extensive homogeneity. Evidence presented here has demonstrated MLSA possesses adequate discriminatory power for outbreak investigation, as well as other potential uses in the molecular epidemiology of leishmaniasis.

With the emergence of analytical software for the inference of viral evolution, a number of studies have focused on estimating important parameters such as the substitution rate and the time to the most recent common ancestor (t MRCA) for rapidly evolving viruses. Coupled with an increasing abundance of sequence data sampled under widely different schemes, an effort to keep results consistent and comparable is needed. This study emphasizes commonly disregarded problems in the inference of evolutionary rates in viral sequence data when sampling is unevenly distributed on a temporal scale through a study of the foot-and-mouth (FMD) disease virus serotypes SAT 1 and SAT 2. Our study shows that clustered temporal sampling in phylogenetic analyses of FMD viruses will strongly bias the inferences of substitution rates and t MRCA because the inferred rates in such data sets reflect a rate closer to the mutation rate rather than the substitution rate. Estimating evolutionary parameters from viral sequences should be performed with due consideration of the differences in short-term and longer-term evolutionary processes occurring within sets of temporally sampled viruses, and studies should carefully consider how samples are combined.

With great interest we read the manuscript entitled “Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agent of Boid Inclusion Body Disease” by Hetzel and colleagues ([1][1]). This very well performed and comprehensive study about the role of divergent

Viruses are the most abundant and the smallest organisms, which are relatively simple to sequence. Genome sequence data of viruses for individual species to populations out‐ number that of other species. Although this offers an opportunity to study viral diversity at varying levels of taxonomic hierarchy, it also poses challenges for systematic and struc‐ tured organization of data and its downstream processing. Extensive computational anal‐ yses using a number of algorithms and programs have opened exciting opportunities for virus discovery and diagnostics, apart from augmenting our understanding of the intri‐ guing world of viruses. Unravelling evolutionary dynamics of viruses permits improved understanding of phenomena such as quasispecies diversity, role of mutations in host switching and drug resistance, which enables the tangible measurements of genotype and phenotype of viruses. Improved understanding of geno-/serotype diversity in corre‐ lation with antigenic diversity will facilitate rational design and development of effica‐ cious vaccines against emerging and re-emerging viruses. Mathematical models developed using the genomic data could be used to predict the spread of viruses due to vector switching and the (re)emergence due to host switching and, thereby, contribute to‐ wards designing public health policies for disease management and control.

Understanding the dynamics of foot‐and‐mouth disease virus (FMDV), an endemic and economically constraining disease, is critical in designing control programmes in Africa. This study investigates the evolutionary epidemiology of SAT1 and SAT2 FMDV in Eastern Africa, as well as between cattle and wild African buffalo. Bayesian phylodynamic models were used to analyse SAT1 and SAT2 VP1 gene segments collected between 1975 and 2016, focusing on the SAT1 and SAT2 viruses currently circulating in Eastern Africa. The root state posterior probabilities inferred from our analyses suggest Zimbabwe as the ancestral location for SAT1 currently circulating in Eastern Africa (p = 0.67). For the SAT2 clade, Kenya is inferred to be the ancestral location for introduction of the virus into other countries in Eastern Africa (p = 0.72). Salient (Bayes factor >10) viral dispersal routes were inferred from Tanzania to Kenya, and from Kenya to Uganda for SAT1 and SAT2, respectively. Results suggest that cattle are the source of the SAT1 and SAT2 clades currently circulating in Eastern Africa. In addition, our results suggest that the majority of SAT1 and SAT2 in livestock come from other livestock rather than wildlife, with limited evidence that buffalo serve as reservoirs for cattle. Insights from the present study highlight the role of cattle movements and anthropogenic activities in shaping the evolutionary history of SAT1 and SAT2 in Eastern Africa. While the results may be affected by inherent limitations of imperfect surveillance, our analysis elucidates the dynamics between host species in this region, which is key to guiding disease intervention activities.

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle.

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ∼60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family.

Turnip mosaic virus (TuMV) infects crops of plant species in the family Brassicaceae worldwide. TuMV isolates were clustered to five lineages corresponding to basal-B, basal-BR, Asian-BR, world-B and OMs. Here, we determined the complete genome sequences of three TuMV basal-BR isolates infecting radish from Shandong and Jilin Provinces in China. Their genomes were all composed of 9833 nucleotides, excluding the 3′-terminal poly(A) tail. They contained two open reading frames (ORFs), with the large one encoding a polyprotein of 3164 amino acids and the small overlapping ORF encoding a PIPO protein of 61 amino acids, which contained the typically conserved motifs found in members of the genus Potyvirus. In pairwise comparison with 30 other TuMV genome sequences, these three isolates shared their highest identities with isolates from Eurasian countries (Germany, Italy, Turkey and China). Recombination analysis showed that the three isolates in this study had no “clear” recombination. The analyses of conserved amino acids changed between groups showed that the codons in the TuMV out group (OGp) and OMs group were the same at three codon sites (852, 1006, 1548), and the other TuMV groups (basal-B, basal-BR, Asian-BR, world-B) were different. This pattern suggests that the codon in the OMs progenitor did not change but that in the other TuMV groups the progenitor sequence did change at divergence. Genetic diversity analyses indicate that the PIPO gene was under the highest selection pressure and the selection pressure on P3N-PIPO and P3 was almost the same. It suggests that most of the selection pressure on P3 was probably imposed through P3N-PIPO.

Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterised as infecting turnips in Iran. The genome of TCTV shares <68 % pairwise identity with other geminiviruses and has a genome organisation similar to that of curtoviruses and topocuvirus. The replication-associated protein (Rep) bears the highest similarity to curtovirus Reps (48.5–69.0 %); however, in the case of the capsid protein (CP), the extent of similarity is only 39.5–44.5 %. We constructed an agroinfectious clone of TCTV and undertook host range studies on ten plant species; in three species (turnip, sugar beet and cowpea), we detected infection which presents curly top symptoms in turnip and sugar beet. The efficiency of TCTV infection in agroinoculated turnip plants was 71.7 %, and the infection was successfully transmitted to 80 % of the healthy turnip plants used in the insect transmission studies by Circulifer haematoceps under greenhouse conditions. We also determined the genome sequence of 14 new TCTV isolates from southern Iran isolated from turnips. We observed ~13 % diversity amongst all the TCTV isolates and found evidence of recombination in the CP- and Rep-coding regions of the genomes.

Transmission lies at the interface of human immunodeficiency virus type 1 (HIV-1) evolution within and among hosts and separates distinct selective pressures that impose differences in both the mode of diversification and the tempo of evolution. In the absence of comprehensive direct comparative analyses of the evolutionary processes at different biological scales, our understanding of how fast within-host HIV-1 evolutionary rates translate to lower rates at the between host level remains incomplete. Here, we address this by analyzing pol and env data from a large HIV-1 subtype C transmission chain for which both the timing and the direction is known for most transmission events. To this purpose, we develop a new transmission model in a Bayesian genealogical inference framework and demonstrate how to constrain the viral evolutionary history to be compatible with the transmission history while simultaneously inferring the within-host evolutionary and population dynamics. We show that accommodating a transmission bottleneck affords the best fit our data, but the sparse within-host HIV-1 sampling prevents accurate quantification of the concomitant loss in genetic diversity. We draw inference under the transmission model to estimate HIV-1 evolutionary rates among epidemiologically-related patients and demonstrate that they lie in between fast intra-host rates and lower rates among epidemiologically unrelated individuals infected with HIV subtype C. Using a new molecular clock approach, we quantify and find support for a lower evolutionary rate along branches that accommodate a transmission event or branches that represent the entire backbone of transmitted lineages in our transmission history. Finally, we recover the rate differences at the different biological scales for both synonymous and non-synonymous substitution rates, which is only compatible with the ‘store and retrieve’ hypothesis positing that viruses stored early in latently infected cells preferentially transmit or establish new infections upon reactivation.

The use of increasingly powerful genotyping tools for the characterisation of pathogens has become a standard component of infectious disease surveillance and outbreak investigations. This thematic issue of Eurosurveillance, published in two parts, provides a series of review and original research articles that gauge progress in molecular epidemiology strategies and tools, and illustrate their applications in public health. Molecular epidemiology of infectious diseases combines traditional epidemiological methods with analysis of genome polymorphisms of pathogens over time, place and person across human populations and relevant reservoirs, to study host–pathogen interactions and infer hypotheses about host-to-host or source-to-host transmission [1-3]. Based on discriminant genotyping of human pathogens, clonally derived strains can be identified as likely links in a chain of transmission [1-3]. In this two-part issue of Eurosurveillance, Goering et al. explain that such biological evidence of clonal linkage complements but does not replace epidemiological evidence of personto-person contact or common exposure to a potential source [3]. Muellner et al. provide clear examples how prediction about infectious disease outcome and transmission risks can be enhanced through integration of pathogen genetic information and epidemiological modelling to inform public health decisions about food-borne disease prevention [4].

The transformation of DNA sequencing technologies has enabled more powerful and comprehensive genetic profiling of microbes. The sheer number of informative loci provided by genome-sequencing allows the investigation of structural variation and horizontal gene transfer as well as delivering novel insights into genetic origins, evolution and epidemiological history. Microbial genomes can be sequenced en masse at high coverage but have associated challenges of high mutation rates and low conservation of genome structure. Consequently, detecting changes in DNA sequences requires a nuanced approach specific to the organism, availability of similar genomes, and types of variation. Here, we outline the high power of genome-sequencing to detect a wide scope of polymorphism classes. Samples without related species on which to scaffold a genome sequence require specific assembly methods that can be enhanced by progressive procedures for improvement. Polymorphism identification depends on genome structure, and error rates in closely related specimens can be reduced by incorporating population-level information. The development of genome analysis platforms is hastening the optimization of variant discovery and has direct applications for pathogen surveillance. Robust variant screening facilitates more sensitive scrutiny of population history, including the origin and emergence of infectious agents, and a deeper understanding of the selective processes that shape microbial phenotypes. Background Microbial genomics is driven by the need to identify molecular markers and genetic switches associated with novel phenotypes. This is most extensively applied to address infectious disease and evolution but also is used for improving food, energy, water and biomolecule production (Suter et al., 2006). The core aim is to link the trait of interest to a defined cellular signature, principally a metabolic or regulatory change. Distinguishing phenotypes at a genetic level provides an enhanced resolution of the molecular events associated with natural variation due to the density of markers. Variability at the level of amino acids in peptides, repetitive DNA copy numbers and individual DNA nucleotides can provide sufficient power for discriminating traits: sample typing protocols have been developed on this basis (Wren, 2000). The most significant limitations for strain profiling are an adequate number of informative markers and the potential to overlook novel variation at other loci (Achtman, 2008). Although the first microbial genome sequence (RNA virus bacteriophage MS2) was completed in 1976 (Fiers et al., 1976), and the era of microbial genomics was proposed to have begun in 1995 (Rasko and Mongodin, 2005) with the bacterial genome of Haemophilus influenzae (Fleischmann et al., 1995), it was the recent development of DNA sequencing technologies more efficient than traditional capillary (Sanger) sequencing that made genome-sequencing accessible (Margulies et al., 2005). Since a 100-fold improvement developed for the Mycoplasma genitalium genome (Margulies et al., 2005), enhancements have continued and Genome SNP Analysis

The silkworm, Bombyx mori, played an important role in the old Silk Road that connected ancient Asia and Europe. However, to date, there have been few studies of the origins and domestication of this species using molecular methods. In this study, DNA sequences of mitochondrial and nuclear loci were used to infer the phylogeny and evolutionary history of the domesticated silkworm and its relatives. All of the phylogenetic analyses indicated a close relationship between the domesticated silkworm and the Chinese wild silkworm. Domestication was estimated to have occurred about 4100 years ago (ya), and the radiation of the different geographic strains of B. mori about 2000 ya. The Chinese wild silkworm and the Japanese wild silkworm split about 23600 ya. These estimates are in good agreement with the fossil evidence and historical records. In addition, we show that the domesticated silkworm experienced a population expansion around 1000 ya. The divergence times and the population dynamics of silkworms presented in this study will be useful for studies of lepidopteran phylogenetics, in the genetic analysis of domestic animals, and for understanding the spread of human civilizations.

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3′ rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

The phytophagous beetle family Curculionidae is the most species‐rich insect family known, with much of this diversity having been attributed to both co‐evolution with food plants and host shifts at key points within the early evolutionary history of the group. Less well understood is the extent to which patterns of host use vary within or among related species, largely because of the technical difficulties associated with quantifying this. Here we develop a recently characterized molecular approach to quantify diet within and between two closely related species of weevil occurring primarily within dry forests on the island of Mauritius. Our aim is to quantify dietary variation across populations and assess adaptive and nonadaptive explanations for this and to characterize the nature of a trophic shift within an ecologically distinct population within one of the species. We find that our study species are polyphagous, consuming a much wider range of plants than would be suggested by the literature. Our data suggest that local diet variation is largely explained by food availability, and locally specialist populations consume food plants that are not phylogenetically novel, but do appear to represent a novel preference. Our results demonstrate the power of molecular methods to unambiguously quantify dietary variation across populations of insect herbivores, providing a valuable approach to understanding trophic interactions within and among local plant and insect herbivore communities.

The genomes of six pigeon paramyxovirus type 1 (PPMV-1) isolated from symptomless pigeons in live poultry markets during the national active surveillance from 2011 to 2013 were sequenced and analyzed in this study. The complete genome lengths of all isolates were 15,192 nucleotides with the gene order of 3’-NP-P-M-F-HN-L-5’. All isolates had the same motif of 112RRQKRF117 at the cleavage site of the fusion protein, which was typical of velogenic Newcastle disease virus (NDV). Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on sequences of complete genomes and six genes revealed that all isolates belonged to genotype VI in class II, but at least 2 sub-genotypes were identified. Most isolates were placed into sub-genotype VIb with the exception of pi/GX/1015/13, which was classified in sub-genotype VIa. The obvious antigenic difference between PPMV-1 isolates and La Sota strain was found based on the R-value calculated by cross hemagglutination inhibition (HI) assay. These results provided the evidence that PPMV-1 could be detected from healthy pigeons, and our study may be useful in designing vaccines used in pigeon, and developing molecular diagnostic tools to monitor and prevent future PPMV-1 outbreaks.