Chris Lauber

Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans

ABSTRACT A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genome was reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C. IMPORTANCE Coronaviruses are capable of infecting humans and many animal species. Most infections caused by human coronaviruses are relatively mild. However, the outbreak of severe acute respiratory syndrome (SARS) caused by SARS-CoV in 2002 to 2003 and the fatal infection of a human by HCoV-EMC/2012 in 2012 show that coronaviruses are able to cause severe, sometimes fatal disease in humans. We have determined the complete genome of HCoV-EMC/2012 using an unbiased virus discovery approach involving next-generation sequencing techniques, which enabled subsequent state-of-the-art bioinformatics, phylogenetics, and taxonomic analyses. By establishing its complete genome sequence, HCoV-EMC/2012 was characterized as a new genotype which is closely related to bat coronaviruses that are distant from SARS-CoV. We expect that this information will be vital to rapid advancement of both clinical and vital research on this emerging pathogen. Coronaviruses are capable of infecting humans and many animal species. Most infections caused by human coronaviruses are relatively mild. However, the outbreak of severe acute respiratory syndrome (SARS) caused by SARS-CoV in 2002 to 2003 and the fatal infection of a human by HCoV-EMC/2012 in 2012 show that coronaviruses are able to cause severe, sometimes fatal disease in humans. We have determined the complete genome of HCoV-EMC/2012 using an unbiased virus discovery approach involving next-generation sequencing techniques, which enabled subsequent state-of-the-art bioinformatics, phylogenetics, and taxonomic analyses. By establishing its complete genome sequence, HCoV-EMC/2012 was characterized as a new genotype which is closely related to bat coronaviruses that are distant from SARS-CoV. We expect that this information will be vital to rapid advancement of both clinical and vital research on this emerging pathogen.

Discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient.

The Polyomaviridae constitute a family of small DNA viruses infecting a variety of hosts. In humans, polyomaviruses can cause infections of the central nervous system, urinary tract, skin, and possibly the respiratory tract. Here we report the identification of a new human polyomavirus in plucked facial spines of a heart transplant patient with trichodysplasia spinulosa, a rare skin disease exclusively seen in immunocompromized patients. The trichodysplasia spinulosa-associated polyomavirus (TSV) genome was amplified through rolling-circle amplification and consists of a 5232-nucleotide circular DNA organized similarly to known polyomaviruses. Two putative “early” (small and large T antigen) and three putative “late” (VP1, VP2, VP3) genes were identified. The TSV large T antigen contains several domains (e.g. J-domain) and motifs (e.g. HPDKGG, pRb family-binding, zinc finger) described for other polyomaviruses and potentially involved in cellular transformation. Phylogenetic analysis revealed a close relationship of TSV with the Bornean orangutan polyomavirus and, more distantly, the Merkel cell polyomavirus that is found integrated in Merkel cell carcinomas of the skin. The presence of TSV in the affected patients skin was confirmed by newly designed quantitative TSV-specific PCR, indicative of a viral load of 105 copies per cell. After topical cidofovir treatment, the lesions largely resolved coinciding with a reduction in TSV load. PCR screening demonstrated a 4% prevalence of TSV in an unrelated group of immunosuppressed transplant recipients without apparent disease. In conclusion, a new human polyomavirus was discovered and identified as the possible cause of trichodysplasia spinulosa in immunocompromized patients. The presence of TSV also in clinically unaffected individuals suggests frequent virus transmission causing subclinical, probably latent infections. Further studies have to reveal the impact of TSV infection in relation to other populations and diseases.

Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Mesoniviridae: a proposed new family in the order Nidovirales formed by a single species of mosquito-borne viruses

Recently, two independent surveillance studies in Côte d’Ivoire and Vietnam, respectively, led to the discovery of two mosquito-borne viruses, Cavally virus and Nam Dinh virus, with genome and proteome properties typical for viruses of the order Nidovirales. Using a state-of-the-art approach, we show that the two insect nidoviruses are (i) sufficiently different from other nidoviruses to represent a new virus family, and (ii) related to each other closely enough to be placed in the same virus species. We propose to name this new family Mesoniviridae. Meso is derived from the Greek word “mesos” (in English “in the middle”) and refers to the distinctive genome size of these insect nidoviruses, which is intermediate between that of the families Arteriviridae and Coronaviridae, while ni is an abbreviation for “nido”. A taxonomic proposal to establish the new family Mesoniviridae, genus Alphamesonivirus, and species Alphamesonivirus 1 has been approved for consideration by the Executive Committee of the ICTV.

Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages

Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged as a leading cause of antibiotic associated diarrheal disease in humans. The genetic basis of the pathogenicity of C. difficile remains poorly understood. In this study we aimed at characterizing the genetic diversity of C. difficile strains by three different methods (PCR ribotyping, multilocus sequence typing and genetic markers) to improve the typing of C. difficile. Our study was performed on a reference collection (Leeds-Leiden/ECDC) of C. difficile PCR ribotype (RT) strains (n=70) expanded with six PCR RT strains highly related to the emerging PCR RTs 027 and 078. Besides PCR ribotyping we used multilocus sequence typing (MLST) using seven housekeeping genes (MLST 7HG) that has recently been developed for characterizing C. difficile isolates as well as analysis of unique genetic markers. Evolutionary relatedness of the sequences determined by MLST 7HG was analyzed in phylogenetic analysis. In total 56 MLST 7HG sequence types (STs) were identified, nine of which were new. Phylogeny reconstruction of the reference set of strains supplemented with the online available C. difficile MLST reference database, revealed six monophyletic lineages of closely related STs. ST-122 (PCR RT131) formed a well-separated branch in the tree and was thus designated as a novel lineage. Furthermore, we confirmed that several PCR RTs are highly related to the emerging PCR RTs 027 and 078 since these types display the same STs (ST-1 and ST-11, respectively). Based on the observed results, we conclude that MLST 7HG is a valuable method to study C. difficile phylogeny.

Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease

ABSTRACT Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

From Stockholm to Malawi: recent developments in studying human polyomaviruses

Until a few years ago the polyomavirus family (Polyomaviridae) included a dozen viruses identified in avian and mammalian hosts. Two of these, the JC and BK-polyomaviruses isolated a long time ago, are known to infect humans and cause severe illness in immunocompromised hosts. Since 2007 an unprecedented number of eight novel polyomaviruses were discovered in humans. Among them are the KI- and WU-polyomaviruses identified in respiratory samples, the Merkel cell polyomavirus found in skin carcinomas and the polyomavirus associated with trichodysplasia spinulosa, a skin disease of transplant patients. Another four novel human polyomaviruses were identified, HPyV6, HPyV7, HPyV9 and the Malawi polyomavirus, so far not associated with any disease. In the same period several novel mammalian polyomaviruses were described. This review summarizes the recent developments in studying the novel human polyomaviruses, and touches upon several aspects of polyomavirus virology, pathogenicity, epidemiology and phylogeny.

The footprint of genome architecture in the largest genome expansion in RNA viruses.

The small size of RNA virus genomes (2-to-32 kb) has been attributed to high mutation rates during replication, which is thought to lack proof-reading. This paradigm is being revisited owing to the discovery of a 3′-to-5′ exoribonuclease (ExoN) in nidoviruses, a monophyletic group of positive-stranded RNA viruses with a conserved genome architecture. ExoN, a homolog of canonical DNA proof-reading enzymes, is exclusively encoded by nidoviruses with genomes larger than 20 kb. All other known non-segmented RNA viruses have smaller genomes. Here we use evolutionary analyses to show that the two- to three-fold expansion of the nidovirus genome was accompanied by a large number of replacements in conserved proteins at a scale comparable to that in the Tree of Life. To unravel common evolutionary patterns in such genetically diverse viruses, we established the relation between genomic regions in nidoviruses in a sequence alignment-free manner. We exploited the conservation of the genome architecture to partition each genome into five non-overlapping regions: 5′ untranslated region (UTR), open reading frame (ORF) 1a, ORF1b, 3′ORFs (encompassing the 3′-proximal ORFs), and 3′ UTR. Each region was analyzed for its contribution to genome size change under different models. The non-linear model statistically outperformed the linear one and captured >92% of data variation. Accordingly, nidovirus genomes were concluded to have reached different points on an expansion trajectory dominated by consecutive increases of ORF1b, ORF1a, and 3′ORFs. Our findings indicate a unidirectional hierarchical relation between these genome regions, which are distinguished by their expression mechanism. In contrast, these regions cooperate bi-directionally on a functional level in the virus life cycle, in which they predominantly control genome replication, genome expression, and virus dissemination, respectively. Collectively, our findings suggest that genome architecture and the associated region-specific division of labor leave a footprint on genome expansion and may limit RNA genome size.

Toward Genetics-Based Virus Taxonomy: Comparative Analysis of a Genetics-Based Classification and the Taxonomy of Picornaviruses

ABSTRACT Virus taxonomy has received little attention from the research community despite its broad relevance. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3890–3904, 2012), we have introduced a quantitative approach to hierarchically classify viruses of a family using pairwise evolutionary distances (PEDs) as a measure of genetic divergence. When applied to the six most conserved proteins of the Picornaviridae, it clustered 1,234 genome sequences in groups at three hierarchical levels (to which we refer as the “GENETIC classification”). In this study, we compare the GENETIC classification with the expert-based picornavirus taxonomy and outline differences in the underlying frameworks regarding the relation of virus groups and genetic diversity that represent, respectively, the structure and content of a classification. To facilitate the analysis, we introduce two novel diagrams. The first connects the genetic diversity of taxa to both the PED distribution and the phylogeny of picornaviruses. The second depicts a classification and the accommodated genetic diversity in a standardized manner. Generally, we found striking agreement between the two classifications on species and genus taxa. A few disagreements concern the species Human rhinovirus A and Human rhinovirus C and the genus Aphthovirus, which were split in the GENETIC classification. Furthermore, we propose a new supergenus level and universal, level-specific PED thresholds, not reached yet by many taxa. Since the species threshold is approached mostly by taxa with large sampling sizes and those infecting multiple hosts, it may represent an upper limit on divergence, beyond which homologous recombination in the six most conserved genes between two picornaviruses might not give viable progeny.

Partitioning the Genetic Diversity of a Virus Family: Approach and Evaluation through a Case Study of Picornaviruses

ABSTRACT The recent advent of genome sequences as the only source available to classify many newly discovered viruses challenges the development of virus taxonomy by expert virologists who traditionally rely on extensive virus characterization. In this proof-of-principle study, we address this issue by presenting a computational approach (DEmARC) to classify viruses of a family into groups at hierarchical levels using a sole criterion—intervirus genetic divergence. To quantify genetic divergence, we used pairwise evolutionary distances (PEDs) estimated by maximum likelihood inference on a multiple alignment of family-wide conserved proteins. PEDs were calculated for all virus pairs, and the resulting distribution was modeled via a mixture of probability density functions. The model enables the quantitative inference of regions of distance discontinuity in the family-wide PED distribution, which define the levels of hierarchy. For each level, a limit on genetic divergence, below which two viruses join the same group, was objectively selected among a set of candidates by minimizing violations of intragroup PEDs to the limit. In a case study, we applied the procedure to hundreds of genome sequences of picornaviruses and extensively evaluated it by modulating four key parameters. It was found that the genetics-based classification largely tolerates variations in virus sampling and multiple alignment construction but is affected by the choice of protein and the measure of genetic divergence. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3905–3915, 2012), we analyze the substantial insight gained with the genetics-based classification approach by comparing it with the expert-based picornavirus taxonomy.