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.
Norovirus (NoV), a member of the Caliciviridae family, is now considered the most common cause of viral gastroenteritis outbreaks in adults worldwide [1]. In the US, NoV has been identified as the cause of over 73% of outbreaks of gastroenteritis [1]. Furthermore, outbreak NoV strains spread rapidly causing great economic burden on society due to medical and social expenses. Consequently, a vaccine or treatment for NoV would be useful in reducing its transmission and alleviating disease symptoms. Our current knowledge of NoV replication and evolution has made it difficult to predict the efficacy of a treatment or longevity of a vaccine, as evidence is emerging that NoV, like many other RNA viruses, exists as a dynamic, rapidly evolving and genetically diverse population [2], [3], [4]. The high level of genetic diversity in RNA viruses is recognised as the basis for their ubiquity and adaptability [5]. Therefore, in order to develop a successful treatment or control program it is first necessary to understand the mechanisms behind NoV replication and evolution. NoV is a small round virion of 27–38 nm in diameter and possesses a single-stranded, positive-sense, polyadenylated, RNA genome of 7400–7700 nucleotides [6]. The human NoV genome is divided into three open reading frames (ORFs). ORF1 encodes for the non-structural proteins, including an NTPase, 3C-like protease and RNA-dependent RNA polymerase (RdRp) [7]. The two structural proteins VP1, the major capsid protein, and VP2, the minor capsid protein are encoded by ORF2 and ORF3, respectively [8], [9]. NoV is a highly diverse genus with up to 61% VP1 amino acid diversity between its five genogroups (GI to GV) [10]. Up to 44% amino acid diversity over VP1 is also observed within the genogroups and has resulted in the further subgrouping of GI, GII and GIII into 8,17 and 2 genotypes, respectively [10]. VP1 exhibits the highest degree of sequence variability in the genome [11], [12]. It consists of three domains, namely the shell (S) domain connected by a flexible hinge (P1 domain) to a protruding domain (P2) [13]. The highly conserved S domain forms the backbone of the capsid structure [13], while the moderately conserved P1 domain encodes the flexible hinge that connects the S and P2 domains. The protruding P2 domain possesses motifs that are involved in binding to the host cell, and hence, the P2 domain is responsible for the antigenicity of the virus [14], [15]. The most clinically significant of the five genogroups is GII, as it is the most prevalent human NoV genogroup detected and more frequently associated with epidemics compared with other genogroups. Of particular interest is GII genotype 4, (GII. 4), because this lineage accounts for 62% of all NoV outbreaks globally [14], [15] and has also caused all five major NoV pandemics in the last decade (1995/1996, U5-95_US strain; 2002, Farmington Hills; 2004, Hunter; 2006,2006a virus; and 2007,2006b virus) [16], [17], [18], [19]. The basis for the increased epidemiological fitness [20] of the GII. 4 strains, as determined by its high incidence and ability to cause pandemics, is currently unknown. Investigations with influenza indicate a link between increased viral evolution and increased viral incidence [21], [22]. However, because of the non-culturable nature of human NoV, variations in rates of evolution have not been calculated for different NoVs and consequently this has not been investigated as a factor in determining viral incidence and epidemiological fitness. Replication efficiency and genetic diversity are both important parameters in viral fitness [23]. The aim of this study was to determine if these two parameters are contributing to the increased epidemiological fitness of the GII. 4 strains. Replication efficiency and genetic diversity are primarily determined by the viral RdRp, as it controls the rate new sequence is introduced into the genome. Therefore using in vitro RdRp assays together with bioinformatics, the replication efficiency, mutation rate and rate of evolution of GII. 4 viruses was compared with other NoV GII genotypes. The results of this study suggest that, like influenza A, the increased incidence of the pandemic GII. 4 lineage may be a result of the combined influence of a high mutation, replication and evolution rate which, together culminate in an increased epidemiological fitness for the GII. 4 strains.
Stool samples containing NoV were obtained from the Department of Microbiology, Prince of Wales Hospital, Sydney, Australia, with the exception of the stool specimen that contained NoV/Mc17/01/Th (GenBank accession numbers AY237413). This stool specimen was obtained from McCormic Hospital, Chiang Mai, Thailand [16]. The six genetically diverse NoV strains used in this study included: three GII. 4 pandemic strains; NoV/Sydney 348/97/AU (of the NoV/US95_96 GII. 4 pandemic lineage) [16], NoV/NZ327/06/NZ (NoV/2006a GII. 4 lineage) [17] and NoV/NSW696T/06/AU (NoV/2006b GII. 4 lineage) [17]. Two recombinant strains; NoV/Sydney C14/02/AU (GII. b ORF1 and GII. 3 ORF2/3 [commonly referred to as GII. b/GII. 3]) [16] and NoV/Sydney4264/01/AU (GII. 4 ORF1 and GII. 10 ORF2/3, [GII. 4/GII. 10]) [16], and a GII. 7 NoV, NoV/Mc17/01/Th associated with rare sporadic cases of gastroenteritis [24]. In this study, the RdRp enzymes are referred to by their genotype, except in the case of the GII. 4 strains, which are referred to by their pandemic name, eg. GII. 4 2006b-RdRp (see Table 1). RdRps from recombinant strains are indicated by an ‘r’ in front of the nomenclature. Viral RNA was extracted from 140 µl of 20% faecal suspension using the QIAmp Viral RNA kit according to manufacturers' instructions (Qiagen, Victoria, Australia). RNA was resuspended in 50 µl of Baxter Steri-pour H2O and stored at −80°C. cDNA synthesis was performed as described previously [16]. The full length capsid gene, P2 domain and RdRp regions were amplified with specific primers (Table 2) using reverse transcriptase - polymerase chain reaction (RT-PCR) methods described in [17]. The amplified RdRp genes were cloned into pGEM-T Easy vector (Promega, Wisconsin, United States). Plasmids and PCR products were purified by PEG precipitation and washed with 70% ethanol. Products were sequenced directly on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, US) using dye-terminator chemistry. pGEM-T Easy vectors containing 1736 bp from the 3′ end of ORF1 were purified using the Quantum prep® plasmid miniprep kit (BioRad, California, United States) and used as template DNA for the construction of expression vectors. Strain specific primers incorporating restriction enzyme sites, were designed to amplify the precise RdRp region of each strain (Table 2). PCR was performed as described previously [17]. PCR products were digested with their corresponding restriction enzymes and cloned into the expression vector pTrcHis2A (Invitrogen, Mount Waverley, Australia). Constructs containing the hepatitis C virus (HCV) genotype 3a RdRp (pVRL69) and HCV genotype 1b RdRp (pVRL75), were used as controls and have been described previously [25]. Site directed mutagenesis of residue 291 in the GII. 4 US95_96-RdRp and the GII. 4 2006a-RdRp was carried out with the Stratagene Quickchange II mutagenesis kit, according to manufacturer' s instructions (Stratagene, La Jolla, United States). The primers used to introduce the mutation into the plasmid are listed in Table 2. The NoV RdRps and control HCV RdRps were expressed in Escherichia coli, as described previously [25], except expression of the NoV RdRps was performed for 4 hr at 30°C. Purity was checked by SDS-PAGE and the identity of the RdRp was confirmed by western blot with an anti-six histidine antibody and peptide sequencing performed by the Bioanalytical Mass Spectrometry Facility (University of New South Wales, Australia). Recombinant RdRp was quantified with a Nanodrop ND-1000 Spectrophotometer (Nanodrop, Wilmington, United States). Kinetic RdRp assays were performed in a final volume of 15 µl and contained 20 mM Tris-HCl (pH 7. 4), 2. 5 mM MnCl2,5 mM DTT, 1 mM EDTA, 500 ng of homopolymeric C RNA template, 2 U RNasin (Promega), 4 mM sodium glutamate and increasing concentrations of [3H]-GTP (Amersham Biosciences, Little Chalfont, UK) ranging from 2 µM to 60 µM. Reactions were initiated with the addition of 50 nM of RdRp and incubated for 9 mins at 25°C. The reactions were terminated by adding EDTA to a final concentration of 60 mM, 10 µg herring sperm DNA and 170 µl of 20% (w/v) trichloroacetic acid. The incorporated radionucleotides were precipitated on ice for 30 min and then filtered through a 96 well GF/C unifilter microplate (Falcon, Franklin Lakes, United States) by a Filtermate harvester (Packard BioSciences, Melbourne, Australia). Using the harvester, the filters were washed thoroughly with water and left to dry. The filter wells were each filled with 25 µl of Microscint scintillation fluid (Packard Biosciences) and radioactivity measured using a Packard liquid scintillation counter (TopCount NXT; Packard Biosciences). Background measurements for each assay consisted of reactions without RdRp and were subtracted from the count per minute (CPM) values obtained for the individual enzyme assays. Results were plotted and statistical analysis performed with the Mann Whitney Test (one-tailed, 95% confidence interval) in GraphPad Prism version 4. 02 (GraphPad Software, San Diego, CA). An in vitro fidelity assay was developed to measure mutation rates and was adapted from Ward et al. [26]. The RdRp assay was performed using conditions described above with a homopolymeric C RNA template, except 82. 1 pmoles of [3H]UTP (2 µCi) or [3H]ATP (4 µCi) (Amersham Biosciences) were added (as the non-complementary nucleotides) with an equimolar amount of GTP (82. 1 pmoles) (Promega) added as the complementary nucleotide. The total amount of ribonucleotide incorporated was calculated in a parallel experiment with the addition of 1 µCi (164. 2 pmoles) [3H]GTP (Amersham Biosciences) as the correct nucleotide. The assay was incubated for 50 min at 25°C. Error frequency of the RdRp was determined by calculating the total number (pmoles) of non-complementary ribonucleotides incorporated and dividing by the total number (pmoles) of [3H]GTP ribonucleotides incorporated. In order to determine the rate of evolution of the rGII. 3, GII. 3, GII. 4 and GII. 7 capsids, the nucleotide sequences of ORF2 were analysed. RNA capsid sequences used for the analysis included eight from this study and 76 sequences from GenBank, with the oldest strains available dating back to 1987. The strains used and their GenBank accession numbers are listed in Text S1. The rate of evolution (substitutions/nucleotide site/year) for GII. 3, GII. b/GII. 3 GII. 4 and GII. 7 NoVs was determined by calculating the number of nucleotide substitutions in ORF2 compared to an ancestral strain and this was plotted against time [27]. The rate of evolution was determined by linear regression with the program GraphPad PRISM® version 4 and was equivalent to the gradient of the line. Pairwise alignments of RNA sequences and evolutionary distances between sequences were carried out using the Maximum Composite Likelihood model in Mega 4. 0 [28]. Bootstrapped trees (1000 data sets) were constructed using the Neighbour-joining method, also with the program Mega 4. 0. In order to determine the amount of selection each genotype is under, the average Ka/Ks ratio was calculated for each genotype' s capsid gene (GII. 4, GII. b/GII. 3 and GII. 7). The Ka/Ks ratio is a measure of nonsynonymous amino acid changes compared to synonymous (silent) changes. Ka/Ks>1 indicates that positive selection is occurring. Ka/Ks = 1 is interpreted as neutral evolution and Ka/Ks<1 is indicative of negative or purifying selection. The program Sliding Windows Alignment Analysis Program (SWAAP) version 1. 0. 2 [29] was utilised. The Nei-Gojobori model was used to calculate Ka and Ks values [30]. The window size was set at 15 bp (5 aa) and the step size was 3 bp (1 aa). Predicted secondary structure analysis of the RdRps and capsid protein VP1 were performed by generating a Protein Data Bank (PDB) file from the amino acid sequence in FastA format using software on the CPHmodels 2. 0 Server [31]. Three dimensional structures were then generated from the PDB files with PyMol [32]. The GenBank accession numbers for the RdRp and capsid genes described in this paper are listed in Text S1.
Over the last decade five NoV pandemics have occurred approximately every two years and all pandemics have been associated with a single NoV genotype, GII. 4 [16], [17], [19], [34]. The reason for the predominance of the GII. 4 strains has been the subject of much speculation but is currently unknown primarily due to a limited understanding of NoV population dynamics and evolution [4], [15], [35]. Studies with other RNA viruses indicate that viral fitness is dependent on many factors, such as, viral mutation, replication efficiency, population size and host factors (reviewed in [2]). To date progress has been made in understanding the role host factors have on NoV prevalence with several studies indicating that variations in viral docking to the blood group antigens may affect infectivity of individuals within a population (reviewed in [36]). In particular, GII. 4 viruses bind to all blood group antigens, whereas, GII. 1 and GII. 3 viruses bind fewer blood group antigens and this could account for higher prevalence of GII. 4 viruses [36]. This paradigm however remains controversial, especially for GII NoV, as not all studies show an association between blood group antigens and clinical infection [37], [38], [39]. Apart from the host/viral interaction, no other factors have been affiliated with NoV fitness. Recent studies performed with poliovirus have shown that an increase in fidelity leads to less genetic diversity and subsequently a reduction in viral fitness and pathogenesis because of a reduced adaptive capacity of the virus [40], [41]. It has been hypothesised that viruses are fitter if they are able to produce a more robust (diverse) population (reviewed in [42], [43], [44]). In the current study we examined whether there was a link between epidemiological fitness, as defined by their incidence, and the rate and accuracy of viral replication. In the present study error rates were assessed directly by examining the mutation rate of the viral RdRp and by analysing the rate of evolution for selected GII lineages. Our results are consistent with mutation rates for the poliovirus RdRp [26] and retrovirus reverse transcriptases [45], which range between 10−3 to 10−5 (Table 1). The more prevalent GII. 4 strains had a 5 to 36-fold higher mutation rate compared to the less frequently detected GII. b/GII. 3 and GII. 7 strains, as determined by in vitro enzyme assays. Consistent with this, the rate of evolution of the capsid was on average 1. 7-fold higher in GII. 4 viruses compared to GII. 3, GII. b/GII. 3 and GII. 7 viruses. The GII. 4 capsids also had a larger Ka/Ks ratio than the GII. b/GII. 3 and GII. 7 strains suggesting that the increased incidence/epidemiological fitness of the GII. 4 strains maybe through greater antigenic drift, a consequence of the higher mutation rate of the GII. 4 RdRp. The mutation rates for the control HCV RdRps (average of 1. 6×10−3 substitutions per nucleotide site, Table 1) were 2-fold higher compared to the GII. 4 RdRps. Evaluation of previously published rates of evolution for the HCV hypervariable region 1 (HVR1) within the envelope 2 glycoprotein (E2) were also higher (6–fold) than the NoV GII. 4 rates of evolution calculated in this study [46] (Table 1). HVR1 was chosen for comparison because, like the NoV capsid gene, it is the most variable region in the genome and under the greatest immune selection. Mutation rate and rate of evolution cannot be directly compared as they are indirectly related due to the increased complexity of evolution in vivo [20]. However, in this study we did find a common trend between the two different measurements of diversity with HCV displaying the highest diversity rate for both measurements compared to NoV. Interestingly, the majority of non-synonymous mutations in the P2 domain for all three NoV genotypes were localised to six common structural sites. These six hypervariable regions within the P2 domain were consistent with hypervariable sites for GII. 4 capsids already identified in other studies [4], [19]. We demonstrated that GII. 7 and GII. 3 viruses shared two and four common hypervariable sites, respectively, with GII. 4 viruses (Fig. 5). Substitutions at one of these sites (residue 395) have been shown to alter GII. 4 strains antigenic profiles [4]. Localization of the hypervariable sites to common regions on the surface of the P2 domain suggests that these regions are likely to be under immune pressure possibly from a neutralizing antibody response [39]. The lower number of amino acid changes at these sites for viruses with a GII. 3 capsid may explain why GII. b/GII. 3 is predominantly associated with gastroenteritis cases in children [47]. This suggests that GII. b/GII. 3 viruses are not as efficient at escaping herd immunity compared to GII. 4 strains and therefore only hosts immunologically naïve to GII. 3 infection are susceptible. Similarly, we propose that the low prevalence of the GII. 7 strain is also a consequence of a low mutation rate in the RdRp resulting in limited antigenic drift and an inability to escape herd immunity. Apart from mutation rate, replication rate is considered to be another major determinant in viral fitness [48]. Replication rates are important because an increased replication rate would produce a larger heterogenous population than a slower replicating virus in the same unit of time, given the same mutation rate. Interestingly, the RdRps from the recent 2006 GII. 4 pandemic strains had a higher nucleotide incorporation rate than the recombinant GII. 4 RdRp and the US95/96-like pandemic GII. 4 RdRp, which could be associated with a point mutation in the RdRp (Thr291Lys). Residue 291 is located in the finger domain, which is comprised of five β sheets that run parallel and strongly interact with each other. The innermost of these five β sheets contains motif F which interacts directly with incoming nucleotides [49]. Therefore, it is plausible that substitutions at residue 291 affects the orientation of motif F due to the strong interaction between the five β sheets and subsequently alters the binding affinity to the incoming nucleotide. Fixation of the Thr291Lys point mutation in the GII. 4 lineage after 2001 has been paralleled with a reduction in the period of stasis between the emergence of new antigenic variants [4]. Alterations in residue 291 after 2001 could have led to an increase in the rate of evolution of GII. 4 strains by increasing the replication rate, however this did not seem to have an effect on mutation rate (Table 1). High replication rates did not always correlate with epidemiological fitness as the NoV strain, GII. 7, had the highest incorporation rate but is considered to be the least fit due to it having the lowest incidence. Therefore, this study suggests mutation rate in combination with a high replication rate are key determinates in epidemiological fitness. Influenza research also indicates a relationship between rate of evolution and epidemiological fitness (reviewed in [21]). New antigenic influenza A variants arise every one to two years and cause more annual epidemics than influenza B, as well as the more devastating pandemics [21]. Once a population has accumulated mass herd immunity to a virus the virus is forced to alter its antigenic determinants, a possibility for viruses with poor fidelity and fast replication rates, or face extinction [50], whereas, viruses such as influenza B, which have higher fidelity and slower antigenic change, are more often associated with sporadic cases [21]. In this study a parallel can be seen in the epidemiology between NoV and influenza, in particular between GII. b/GII. 3 viruses and influenza B and GII. 4 viruses and influenza A. In summary, this study supports the hypothesis that epidemiological fitness is a consequence of the ability of the virus to generate genetic diversity, as the NoV pandemic GII. 4 strains were associated with an increased replication and mutation rate. Therefore, it would seem that GII. 4 viruses, as opposed to GII. b/GII. 3 and GII. 7 viruses, have reached a balance in their replication rate and mutation rate that is better suited to viral adaptation. In contrast, it would seem that the GII. 7 lineage, despite having a high replication rate, has a low mutation rate that limits its adaptation and therefore its incidence. It is important to improve our understanding of the mechanisms underlying NoV epidemiological fitness as future pandemics are expected.
Since 1995, norovirus has caused five pandemics of acute gastroenteritis. These pandemics spread across the globe within a few months, causing great economic burden on society due to medical and social expenses. Norovirus, like influenza virus, has over 40 genotypes circulating within the population at the same time. However, it is only a single genotype, known as genogroup II genotype 4 (GII. 4), that causes mass outbreaks and pandemics. Very little research has been conducted to determine why GII. 4 viruses can cause pandemics. Consequently, we compared the evolution properties of several pandemic GII. 4 strains to non-pandemic strains and found that the GII. 4 viruses were undergoing evolution at a much higher rate than the non-pandemic norovirus strains. This phenomenon is similar to influenza virus, where an increase in antigenic drift has been associated with increased outbreaks. This discovery has important implications in understanding norovirus incidence and also the development of a vaccine and treatment for norovirus.
virology/mechanisms of resistance and susceptibility, including host genetics
virology/virus evolution and symbiosis
virology/emerging viral diseases
Rapid Evolution of Pandemic Noroviruses of the GII.4 Lineage