John-Sebastian Eden

Rapid Evolution of Pandemic Noroviruses of the GII.4 Lineage

Over the last fifteen years there have been five pandemics of norovirus (NoV) associated gastroenteritis, and the period of stasis between each pandemic has been progressively shortening. NoV is classified into five genogroups, which can be further classified into 25 or more different human NoV genotypes; however, only one, genogroup II genotype 4 (GII.4), is associated with pandemics. Hence, GII.4 viruses have both a higher frequency in the host population and greater epidemiological fitness. The aim of this study was to investigate if the accuracy and rate of replication are contributing to the increased epidemiological fitness of the GII.4 strains. The replication and mutation rates were determined using in vitro RNA dependent RNA polymerase (RdRp) assays, and rates of evolution were determined by bioinformatics. GII.4 strains were compared to the second most reported genotype, recombinant GII.b/GII.3, the rarely detected GII.3 and GII.7 and as a control, hepatitis C virus (HCV). The predominant GII.4 strains had a higher mutation rate and rate of evolution compared to the less frequently detected GII.b, GII.3 and GII.7 strains. Furthermore, the GII.4 lineage had on average a 1.7-fold higher rate of evolution within the capsid sequence and a greater number of non-synonymous changes compared to other NoVs, supporting the theory that it is undergoing antigenic drift at a faster rate. Interestingly, the non-synonymous mutations for all three NoV genotypes were localised to common structural residues in the capsid, indicating that these sites are likely to be under immune selection. This study supports the hypothesis that the ability of the virus to generate genetic diversity is vital for viral fitness.

Redefining the invertebrate RNA virosphere

Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Recombination within the Pandemic Norovirus GII.4 Lineage

ABSTRACT Norovirus (NoV) is the leading cause of viral gastroenteritis globally. Since 1996, NoV variants of a single genetic lineage, GII.4, have been associated with at least six pandemics of acute gastroenteritis and caused between 62 and 80% of all NoV outbreaks. The emergence of these novel GII.4 variants has been attributed to rapid evolution and antigenic variation in response to herd immunity; however, the contribution of recombination as a mechanism facilitating emergence is increasingly evident. In this study, we sought to examine the role that intragenotype recombination has played in the emergence of GII.4 variants. Using a genome-wide approach including 25 complete genome sequences generated as part of this study, 11 breakpoints were identified within the NoV GII.4 lineage. The breakpoints were located at three recombination hot spots: near the open reading frame 1/2 (ORF1/2) and ORF2/3 overlaps, as well as within ORF2, which encodes the viral capsid, at the junction of the shell and protruding domains. Importantly, we show that recombination contributed to the emergence of the recent pandemic GII.4 variant, New Orleans 2009, and a newly identified GII.4 variant, termed Sydney 2012. Reconstructing the evolutionary history of the GII.4 lineage reveals the widespread impact of both inter- and intragenotype recombination on the emergence of many GII.4 variants. Lastly, this study highlights the many challenges in the identification of true recombination events and proposes that guidelines be applied for identifying NoV recombinants.

Contribution of Intra- and Interhost Dynamics to Norovirus Evolution

ABSTRACT Norovirus (NoV) is an emerging RNA virus that has been associated with global epidemics of gastroenteritis. Each global epidemic arises with the emergence of novel antigenic variants. While the majority of NoV infections are mild and self-limiting, in the young, elderly, and immunocompromised, severe and prolonged illness can result. As yet, there is no vaccine or therapeutic treatment to prevent or control infection. In order to design effective control strategies, it is important to understand the mechanisms and source of the new antigenic variants. In this study, we used next-generation sequencing (NGS) technology to investigate genetic diversification in three contexts: the impact of a NoV transmission event on viral diversity and the contribution to diversity of intrahost evolution over both a short period of time (10 days), in accordance with a typical acute NoV infection, and a prolonged period of time (288 days), as observed for NoV chronic infections of immunocompromised individuals. Investigations of the transmission event revealed that minor variants at frequencies as low as 0.01% were successfully transmitted, indicating that transmission is an important source of diversity at the interhost level of NoV evolution. Our results also suggest that chronically infected immunocompromised subjects represent a potential reservoir for the emergence of new viral variants. In contrast, in a typical acute NoV infection, the viral population was highly homogenous and relatively stable. These results indicate that the evolution of NoV occurs through multiple mechanisms.

The emergence and evolution of the novel epidemic norovirus GII.4 variant Sydney 2012.

Norovirus is the leading cause of acute gastroenteritis with most infections caused by GII.4 variants. To understand the evolutionary processes that contribute to the emergence of GII.4 variants, we examined the molecular epidemiology of norovirus-associated acute gastroenteritis in Australia and New Zealand from 893 outbreaks between 2009 and 2012. Throughout the study GII.4 New Orleans 2009 was predominant; however, during 2012 it was replaced by an emergent GII.4 variant, Sydney 2012. An evolutionary analysis of capsid gene sequences was performed to determine the origins and selective pressures driving the emergence of these recently circulating GII.4 variants. This revealed that both New Orleans 2009 and Sydney 2012 share a common ancestor with GII.4 Apeldoorn 2007. Furthermore, pre-epidemic ancestral variants of each virus were identified up to two years before their pandemic emergence. Adaptive changes at known blockade epitopes in the viral capsid were also identified that likely contributed to their emergence.

Nonnucleoside Inhibitors of Norovirus RNA Polymerase: Scaffolds for Rational Drug Design

ABSTRACT Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide, causing over 200,000 deaths a year. NoV is nonenveloped, with a single-stranded RNA genome, and is primarily transmitted person to person. The viral RNA-dependent RNA polymerase (RdRp) is critical for the production of genomic and subgenomic RNA and is therefore a prime target for antiviral therapies. Using high-throughput screening, nearly 20,000 “lead-like” compounds were tested for inhibitory activity against the NoV genogroup II, genotype 4 (GII.4) RdRp. The four most potent hits demonstrated half-maximal inhibitory concentrations (IC50s) between 5.0 μM and 9.8 μM against the target RdRp. Compounds NIC02 and NIC04 revealed a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 RdRp inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious GV.1 murine norovirus (MNV). NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with 50% effective concentrations (EC50s) of 30.1 μM and 71.1 μM, respectively, while NIC10 and NIC12 had no observable effect on the NoV GI.1 replicon. In the MNV model, NIC02 reduced plaque numbers, size, and viral RNA levels in a dose-dependent manner (EC50s between 2.3 μM and 4.8 μM). The remaining three compounds also reduced MNV replication, although with higher EC50s, ranging from 32 μM to 38 μM. In summary, we have identified novel nonnucleoside inhibitor scaffolds that will provide a starting framework for the development and future optimization of targeted antivirals against NoV.

The evolutionary history of vertebrate RNA viruses

Our understanding of the diversity and evolution of vertebrate RNA viruses is largely limited to those found in mammalian and avian hosts and associated with overt disease. Here, using a large-scale meta-transcriptomic approach, we discover 214 vertebrate-associated viruses in reptiles, amphibians, lungfish, ray-finned fish, cartilaginous fish and jawless fish. The newly discovered viruses appear in every family or genus of RNA virus associated with vertebrate infection, including those containing human pathogens such as influenza virus, the Arenaviridae and Filoviridae families, and have branching orders that broadly reflected the phylogenetic history of their hosts. We establish a long evolutionary history for most groups of vertebrate RNA virus, and support this by evaluating evolutionary timescales using dated orthologous endogenous virus elements. We also identify new vertebrate-specific RNA viruses and genome architectures, and re-evaluate the evolution of vector-borne RNA viruses. In summary, this study reveals diverse virus–host associations across the entire evolutionary history of the vertebrates.Around 200 new vertebrate-specific viruses are discovered, and every vertebrate-specific viral family known to infect mammals and birds is also present in amphibians, reptiles or fish, suggesting that evolution of vertebrate viruses mirrors that of vertebrate hosts.

Adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of Middle East respiratory syndrome coronavirus

BackgroundThe newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) that first appeared in Saudi Arabia during the summer of 2012 has to date (20th September 2013) caused 58 human deaths. MERS-CoV utilizes the dipeptidyl peptidase 4 (DPP4) host cell receptor, and analysis of the long-term interaction between virus and receptor provides key information on the evolutionary events that lead to the viral emergence.FindingsWe show that bat DPP4 genes have been subject to significant adaptive evolution, suggestive of a long-term arms-race between bats and MERS related CoVs. In particular, we identify three positively selected residues in DPP4 that directly interact with the viral surface glycoprotein.ConclusionsOur study suggests that the evolutionary lineage leading to MERS-CoV may have circulated in bats for a substantial time period.

Molecular epidemiology of norovirus in Singapore, 2004–2011

Norovirus (NoV) is the most common cause of sporadic and epidemic gastroenteritis, globally. This study aimed to investigate the molecular epidemiology of NoV‐associated acute gastroenteritis in Singapore by classifying circulating NoV genotypes and genogroup II, genotype 4 (GII.4) variants between September 2004 and February 2011. The temporal dominance and antigenic variation within the circulating epidemic NoV GII.4 variants was also examined, in order to compare the trends in Singapore to those observed globally during the same period. A total of 312 of 1,060 fecal specimens were positive for NoV RNA, using a quantitative RT‐PCR. In a subset (125 of 312) of NoV positive samples, the 5′ end of ORF2 (region C) of the GI or GII NoV genome was amplified and sequenced. Subsequent phylogenetic analysis identified GII.4 was the most commonly identified genotype representing 80.8% (101/125) of NoV sequenced in this study. The predominant GII.4 variants in circulation during the 2004–2011 epidemic periods were Hunter 2004 (2004–2005), Den Haag 2006b (2006–2009), and New Orleans 2009 (2009–2011). Amino acid variation within the P2 domain of the major capsid protein, VP1, was followed longitudinally within the GII.4 lineage. A constant turnover of variant‐specific amino acid change was observed, particularly within the antigenic epitopes A, C and E. In conclusion, this study has characterized the NoV strains in circulation in Singapore between 2004 and 2011. The molecular epidemiology and persistence of GII.4 pandemic NoV lineages in Singapore was similar to trends seen globally, with a noted absence of the Asia 2003 variant. J Med. Virol. 85:1842–1851, 2013.

A Multi-Site Study of Norovirus Molecular Epidemiology in Australia and New Zealand, 2013-2014

Background Norovirus (NoV) is the major cause of acute gastroenteritis across all age groups. In particular, variants of genogroup II, genotype 4 (GII.4) have been associated with epidemics globally, occurring approximately every three years. The pandemic GII.4 variant, Sydney 2012, was first reported in early 2012 and soon became the predominant circulating NoV strain globally. Despite its broad impact, both clinically and economically, our understanding of the fundamental diversity and mechanisms by which new NoV strains emerge remains limited. In this study, we describe the molecular epidemiological trends of NoV-associated acute gastroenteritis in Australia and New Zealand between January 2013 and June 2014. Methodology Overall, 647 NoV-positive clinical faecal samples from 409 outbreaks and 238 unlinked cases of acute gastroenteritis were examined by RT-PCR and sequencing. Phylogenetic analysis was then performed to identify NoV capsid genotypes and to establish the temporal dominance of circulating pandemic GII.4 variants. Recombinant viruses were also identified based on analysis of the ORF1/2 overlapping region. Findings Peaks in NoV activity were observed, however the timing of these epidemics varied between different regions. Overall, GII.4 NoVs were the dominant cause of both outbreaks and cases of NoV-associated acute gastroenteritis (63.1%, n = 408/647), with Sydney 2012 being the most common GII.4 variant identified (98.8%, n = 403/408). Of the 409 reported NoV outbreaks, aged-care facilities were the most common setting in both Western Australia (87%, n = 20/23) and New Zealand (58.1%, n = 200/344) while most of the NoV outbreaks were reported from hospitals (38%, n = 16/42) in New South Wales, Australia. An analysis of a subset of non-GII.4 viruses from all locations (125/239) showed the majority (56.8%, n = 71/125) were inter-genotype recombinants. These recombinants were surprisingly diverse and could be classified into 18 distinct recombinant types, with GII.P16/GII.13 (24% of recombinants) the most common. Conclusion This study revealed that following its emergence in 2012, GII.4 Sydney 2012 variant continued to be the predominant cause of NoV-associated acute gastroenteritis in Australia and New Zealand between 2013 and 2014.