The evolutionary history of hepaciviruses.

In the search for natural reservoirs of hepatitis C virus (HCV), a broad diversity of non-human viruses within the Hepacivirus genus has been uncovered. However, the evolutionary dynamics that shaped the diversity and timescale of hepaciviruses evolution remain elusive. To gain further insights into the origins and evolution of this genus, we screened a large dataset of wild mammal samples (n = 1,672) from Africa and Asia, and generated 34 full-length hepacivirus genomes. Phylogenetic analysis of these data together with publicly available genomes emphasizes the importance of rodents as hepacivirus hosts and we identify 13 rodent species and 3 rodent genera (in Cricetidae and Muridae families) as novel hosts of hepaciviruses. Through co-phylogenetic analyses, we demonstrate that hepacivirus diversity has been affected by cross-species transmission events against the backdrop of detectable signal of virus-host co-divergence in the deep evolutionary history. Using a Bayesian phylogenetic multidimensional scaling approach, we explore the extent to which host relatedness and geographic distances have structured present-day hepacivirus diversity. Our results provide evidence for a substantial structuring of mammalian hepacivirus diversity by host as well as geography, with a somewhat more irregular diffusion process in geographic space. Finally, using a mechanistic model that accounts for substitution saturation, we provide the first formal estimates of the timescale of hepacivirus evolution and estimate the origin of the genus to be about 22 million years ago. Our results offer a comprehensive overview of the micro- and macroevolutionary processes that have shaped hepacivirus diversity and enhance our understanding of the long-term evolution of the Hepacivirus genus.

Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak.

The 2018 Nigerian Lassa fever season saw the largest ever recorded upsurge of cases, raising concerns over the emergence of a strain with increased transmission rate. To understand the molecular epidemiology of this upsurge, we performed, for the first time at the epicenter of an unfolding outbreak, metagenomic nanopore sequencing directly from patient samples, an approach dictated by the highly variable genome of the target pathogen. Genomic data and phylogenetic reconstructions were communicated immediately to Nigerian authorities and the World Health Organization to inform the public health response. Real-time analysis of 36 genomes and subsequent confirmation using all 120 samples sequenced in the country of origin revealed extensive diversity and phylogenetic intermingling with strains from previous years, suggesting independent zoonotic transmission events and thus allaying concerns of an emergent strain or extensive human-to-human transmission.

Erratum: Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

[This corrects the article DOI: 10.1093/ve/vey027.][This corrects the article DOI: 10.1093/ve/vey027.].

Whole genome analysis of local Kenyan and global sequences unravels the epidemiological and molecular evolutionary dynamics of RSV genotype ON1 strains.

The respiratory syncytial virus (RSV) group A variant with the 72-nucleotide duplication in the G gene, genotype ON1, was first detected in Kilifi in 2012 and has almost completely replaced circulating genotype GA2 strains. This replacement suggests some fitness advantage of ON1 over the GA2 viruses in Kilifi, and might be accompanied by important genomic substitutions in ON1 viruses. Close observation of such a new virus genotype introduction over time provides an opportunity to better understand the transmission and evolutionary dynamics of the pathogen. We have generated and analysed 184 RSV-A whole-genome sequences (WGSs) from Kilifi (Kenya) collected between 2011 and 2016, the first ON1 genomes from Africa and the largest collection globally from a single location. Phylogenetic analysis indicates that RSV-A circulation in this coastal Kenya location is characterized by multiple introductions of viral lineages from diverse origins but with varied success in local transmission. We identified signature amino acid substitutions between ON1 and GA2 viruses' surface proteins (G and F), polymerase (L), and matrix M2-1 proteins, some of which were positively selected, and thereby provide an enhanced picture of RSV-A diversity. Furthermore, five of the eleven RSV open reading frames (ORFs) (G, F, L, N, and P) formed distinct phylogenetic clusters for the two genotypes. This might suggest that coding regions outside of the most frequently studied G ORF also play a role in the adaptation of RSV to host populations, with the alternative possibility that some of the substitutions are neutral and provide no selective advantage. Our analysis provides insight into the epidemiological processes that define RSV spread, highlights the genetic substitutions that characterize emerging strains, and demonstrates the utility of large-scale WGS in molecular epidemiological studies.

The multi-faceted dynamics of HIV-1 transmission in Northern Alberta: A combined analysis of virus genetic and public health data.

Molecular epidemiology has become a key tool for tracking infectious disease epidemics. Here, the spread of the most prevalent HIV-1 subtypes in Northern Alberta, Canada, was characterized with a Bayesian phylogenetic approach using 1146 HIV-1 pol sequences collected between 2007 and 2013 for routine clinical management purposes. Available patient metadata were qualitatively interpreted and correlated with onwards transmission using Fisher exact tests and logistic regression. Most infections were from subtypes A (n=36), B (n=815) and C (n=211). Africa is the dominant origin location for subtypes A and C while the subtype B epidemic was seeded from the USA and Middle America and, from the early 1990s onwards, mostly by interprovincial spread. Subtypes A (77.8%) and C (74.0%) were usually heterosexually transmitted and circulate predominantly among Blacks (61.1% and 85% respectively). Subtype B was mostly found among Caucasians (48.6%) and First Nations (36.8%), and its modes of transmission were stratified by ethnic origin. Compared to subtypes A (5.6%) and C (3.8-10.0%), a larger portion of subtype B patients were found within putative provincial transmission networks (20.3-29.5%), and this almost doubled when focusing on nationwide transmission clusters (37.9-57.5%). No clear association between cluster membership and particular patient characteristics was found. This study reveals complex and multi-faceted transmission dynamics of the HIV-1 epidemic in this otherwise low HIV prevalence population in Northern Alberta, Canada. These findings can aid public health planning.

Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts.

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

HBV subgenotype misclassification expands quasi-subgenotype A3.

Recently, we proposed a new classification for 'subgenotype A' of hepatitis B virus (HBV), in which the novel 'quasi-subgenotype A3' group comprising HBV 'subgenotype A3', 'tentative A4', and A5 was introduced. Newly 'Tentative subgenotype A7' strains from Cameroon were introduced by Hubschen et al. However, our meticulous phylogenetic analysis demonstrated that these isolates should also be classified into 'quasi-subgenotype A3'. Such misclassification can be avoided by following established principles for HBV subgenotyping. Moreover, their close evolutionary relationship with A3 highlights our hypothesis that geographical origin may be an important factor in further classification of HBV subgenotypes.

HCV genotype distribution in Flanders and Brussels (Belgium): unravelling the spread of an uncommon HCV genotype 5a cluster.

In order to study the hepatitis C virus (HCV) epidemiology in Flanders, Belgium, the HCV genotype of 2,301 patients diagnosed with HCV between 2001 and 2009 was determined. HCV genotyping was conducted using the Versant LiPA 1.0 or Versant LiPA 2.0 assay. To explore the transmission history of a remarkable cluster of the rarely found HCV genotype 5a, face-to-face interviews based on detailed questionnaires and maximum likelihood phylogenetic analysis were performed. HCV genotype 1 was the most prevalent genotype in all provinces, followed by HCV genotype 3 in East Flanders, Antwerp, Flemish Brabant and Limburg. In Brussels, HCV genotype 4 was the second most prevalent genotype. This observation is due to the immigration of patients from the Middle East and Africa. Remarkably, a cluster of HCV genotype 5a was found in West Flanders, where it represents the second most prevalent genotype, accounting for 26.2% of HCV infections. We could not identify one major transmission source explaining the whole HCV genotype 5a epidemic. Instead, several smaller possible transmission chains were identified and confirmed phylogenetically. Overall, the HCV genotype 5a epidemic in West Flanders seems to be mainly associated with blood transfusion and unsafe medical practices.

The epidemic history of hepatitis C among injecting drug users in Flanders, Belgium.

We employed recently developed statistical methods to explore the epidemic behaviour of hepatitis C subtype 1a and subtype 3a among injecting drug users (IDUs) in Flanders, Belgium, using new gene sequence data sampled among two geographically distinct populations of IDUs. First the extent of hepatitis C transmission across regions/countries was studied through calculation of association indices. It was shown that viral exchange had occurred between both populations in Flanders as well as across international borders. Furthermore, evidence was found suggestive of subtypes 1a and 3a predominantly circulating in subpopulations of Flemish IDUs, exhibiting different degrees of travelling/migration behaviour. Secondly, through coalescent-based analysis the viral epidemic history of the hepatitis C subtype 1a and 3a epidemics was inferred. Evidence was found for different dynamic forces driving both epidemics. Moreover, results suggested that the hepatitis C subtype 3a epidemic has reached a steady state, while the hepatitis C 1a epidemic has not, which therefore might become the predominant subtype among IDUs.

Evidence for a complex mosaic genome pattern in a full-length hepatitis C virus sequence.

The genome of the hepatitis C virus (HCV) exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061) that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a "simple" HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full-length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.

Estimation of an in vivo fitness landscape experienced by HIV-1 under drug selective pressure useful for prediction of drug resistance evolution during treatment.

MOTIVATION: HIV-1 antiviral resistance is a major cause of antiviral treatment failure. The in vivo fitness landscape experienced by the virus in presence of treatment could in principle be used to determine both the susceptibility of the virus to the treatment and the genetic barrier to resistance. We propose a method to estimate this fitness landscape from cross-sectional clinical genetic sequence data of different subtypes, by reverse engineering the required selective pressure for HIV-1 sequences obtained from treatment naive patients, to evolve towards sequences obtained from treated patients. The method was evaluated for recovering 10 random fictive selective pressures in simulation experiments, and for modeling the selective pressure under treatment with the protease inhibitor nelfinavir. RESULTS: The estimated fitness function under nelfinavir treatment considered fitness contributions of 114 mutations at 48 sites. Estimated fitness correlated significantly with the in vitro resistance phenotype in 519 matched genotype-phenotype pairs (R(2) = 0.47 (0.41 - 0.54)) and variation in predicted evolution under nelfinavir selective pressure correlated significantly with observed in vivo evolution during nelfinavir treatment for 39 mutations (with FDR = 0.05). AVAILABILITY: The software is available on request from the authors, and data sets are available from http://jose.med.kuleuven.be/~kdforc0/nfv-fitness-data/.

SlidingBayes: exploring recombination using a sliding window approach based on Bayesian phylogenetic inference.

We developed a software tool (SlidingBayes) for recombination analysis based on Bayesian phylogenetic inference. Sliding-Bayes provides a powerful approach for detecting potential recombination, especially between highly divergent sequences and complex HIV-1 recombinants for which simpler methods like neighbor joining (NJ) may be less powerful. SlidingBayes guides Markov Chain Monte Carlo (MCMC) sampling performed by MrBayes in a sliding window across the alignment (Bayesian scanning). The tool can be used for nucleotide and amino acid sequences and combines all the modeling possibilities of MrBayes with the ability to plot the posterior probability support for clustering of various combinations of taxa.

Cyclorraphan yolk proteins and lepidopteran minor yolk proteins originate from two unrelated lipase families.

Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins.

Analysis of the evolutionary relationships of HIV-1 and SIVcpz sequences using bayesian inference: implications for the origin of HIV-1.

The most plausible origin of HIV-1 group M is an SIV lineage currently represented by SIVcpz isolated from the chimpanzee subspecies Pan troglodytes troglodytes. The origin of HIV-1 group O is less clear. Putative recombination between any of the HIV-1 and SIVcpz sequences was tested using bootscanning and Bayesian-scanning plots, as well as a new method using a Bayesian multiple change-point (BMCP) model to infer parental sequences and crossing-over points. We found that in the case of highly divergent sequences, such as HIV-1/SIVcpz, Bayesian scanning and BMCP methods are more appropriate than bootscanning analysis to investigate spatial phylogenetic variation, including estimating the boundaries of the regions with discordant evolutionary relationships and the levels of support of the phylogenetic clusters under study. According to the Bayesian scanning plots and BMCP method, there was strong evidence for discordant phylogenetic clustering throughout the genome: (1) HIV-1 group O clustered with SIVcpzANT/TAN in middle pol, and partial vif/env; (2) SIVcpzGab1 clustered with SIVcpzANT/TAN in 3'pol/vif, and middle env; (3) HIV-1 group O grouped with SIVcpzCamUS and SIVcpzGab1 in p17/p24; (4) HIV-1 group M was more closely related to SIVcpzCamUS in 3'gag/pol and in middle pol, whereas in partial gp120 group M clustered with group O. Conditionally independent phylogenetic analysis inferred by maximum likelihood (ML) and Bayesian methods further confirmed these findings. The discordant phylogenetic relationships between the HIV-1/SIVcpz sequences may have been caused by ancient recombination events, but they are also due, at least in part, to altered rates of evolution between parental SIVcpz lineages.

Mannan-binding lectin (MBL) gene polymorphisms in ulcerative colitis and Crohn's disease.

The inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC), are complex multifactorial traits involving both environmental and genetic factors. Mannan-binding lectin (MBL) plays an important role in non-specific immunity and complement activation. Point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene are associated with decreased MBL plasma concentrations and increased susceptibility to various infectious diseases. If these MBL mutations could lead to susceptibility to putative IBD-etiological microbial agents, or could temper the complement-mediated mucosal damage in IBD, MBL could function as the link between certain microbial, immunological and genetic factors in IBD. In this study, we investigated the presence of the codon 52, 54 and 57 mutations of the MBL gene in 431 unrelated IBD patients, 112 affected and 141 unaffected first-degree relatives, and 308 healthy control individuals. In the group of sporadic IBD patients (n = 340), the frequency of the investigated MBL variants was significantly lower in UC patients when compared with CD patients (P = 0.01) and with controls (P = 0.02). These results suggest that MBL mutations which decrease the formation of functional MBL could protect against the clinical development of sporadic UC, but not of CD. This could be explained by the differential T-helper response in both diseases.