Genogroup

Reference strains (n = 91) for all serotypes (excluding 6D) were acquired from Statens Serum Institut. Reference strain for 6D was kindly provided by The National Institute for Health and Welfare (THL), Finland. A total of 926 clinical isolates were selected from the archives of the Public Health England (PHE) National Reference Lab as a test cohort; for serotypes found to belong to a genogroup, at least 10 isolates were selected where available. Post genomic-sequence data cleansing (to remove repeat isolates from the same patient, mixed cultures, other species and MLST partial profiles or failures) resulted in 871 isolates (Development Set in Table 1). In addition, 2079 prospective or research-related isolates were sequenced as part of the UK validation cohort. This cohort covers 72 of the commonly circulating serotypes (including all vaccine serotypes), and includes prospective isolates received by PHE during 2015, isolates selected as part of research projects and epidemiological investigations (15A (n = 196) and 19A (n = 249), respectively) and archived isolates for rarer serotypes. Post genomic-sequence data cleansing of this dataset resulted in a total of 2065 isolates (Validation Set in Table 1). All isolates were serotyped on receipt as part of the PHE enhanced surveillance programme using slide agglutination with Statens Serum Institut typing sera.
Genomic data for non-UK isolates were obtained from Streptococcus pneumoniae isolate database hosted in BIGSdb (http://pubmlst.org/software/database/bigsdb/) (Jolley & Maiden, 2010 (link)) and the European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena). Specifically, three collections were used; a set of 2531 isolates from Thailand initially described by Chewapreecha et al. (2014) (link), an Icelandic panel of 252 serogroup 6 isolates described in Van Tonder et al. (2015) (link) and a USA panel of 181 invasive isolates available in ENA as study SRP059723.

Coxiella burnetii isolates used in this study and their associated epidemiological data are listed in Table S2. As 18 of our 63 isolates overlapped with isolates used by Glazunova et al. [7] (link), we were able to compare and evaluate the consistency of the results. Additionally, 21 of our isolates overlapped with those used by Hendrix et al. [10] (link) who describe genomic groups defined by restriction enzyme banding patterns. This overlap allowed us to predict genomic groups based on our phylogeny.
Genomic DNA was isolated using the QiaAmp DNA Mini Kit (Qiagen, Valencia, CA, USA), following the tissue lysis protocol with proteinase K lysis performed at 56°C overnight. For the 12 melt-MAMA assays, 1 µL of DNA was used in a total PCR reaction volume of 10 µL that contained 1× SYBR® Green PCR Master Mix (Applied Biosystems by Life Technologies, Foster City, CA, USA), 300 nM consensus primer, and variable amounts of allele-specific primers (see Table 1). Thermal cycling conditions were: 50°C for 2 min., 95°C for 10 min., followed by the specified number of cycles (see Table 1) of 95°C for 15 sec., 55°C for 1 min. and concluding with a dissociation stage of 95°C for 15 sec., 55°C for 15 sec., 95°C for 15 sec. Analysis of melt curves were performed as described by Vogler et al. [14] (link). For select samples from each genogroup, results obtained by Melt-MAMA assays were confirmed by MST of entire loci as described by Glazunova et al. [7] (link) with the exclusion of using plasmid vectors for cloning and amplification.
For the two TaqMan minor-groove binding dual-probe assays, 1 µL of DNA was also used in a total reaction volume of 10 µL that contained 1× TaqMan® Genotyping Master Mix (Applied Biosystems by Life Technologies, Foster City, CA, USA), 900 nM of each primer and 200 nM of each probe (Table 2). Thermal cycling conditions were: 50°C for 2 min., 95°C for 10 min., followed by 40 cycles of 95°C for 15 sec., 60°C for 1 min. Results were analyzed as described by Easterday et al. [16] (link). All assays were run on an Applied Biosystems 7900HT Fast real-time PCR system with SDS v2.3 or v2.4 software. MST genotype designation(s) and phylogenetic group predictions were based on the results from the 14 SNP assays.

An EVA71 reverse genetics system was generated from genogroup B2 strain MS/7423/87. Viral RNA was extracted from supernatant samples, and the cDNA sequence was reverse transcribed using the Transcriptor first-strand cDNA synthesis kit (Roche, Switzerland). DNA was amplified using sequence-specific primers, and the amplicon was introduced under a T7 promoter and downstream of a hammerhead ribozyme. HeLa cells were obtained from the National Institute for Biological Standards and Control (NIBSC), and Vero cells were obtained from ATCC. PichiaPink strain 1 was purchased from Invitrogen (USA).

All culture-positive isolates were further characterised using WGS on the Illumina platform to define genogroup, MLST lineages and genetic similarities, as previously described [25 (link)]. However, from each culture-positive sample only one colony was characterised.
A phylogenetic tree of carriage isolates was created with BIGSdb integrated in the Neisseria PubMLST database [26 (link)] using the genome comparator tool (based on 1605 N. meningitidis core loci) and visualised with the online tool iTOL [27 (link)].
N. meningitidis detected by PCR alone, and where culture isolate was lacking, were genogrouped using multiplex real-time PCR for the groups A, B, C, W, Y and X according to the instructions provided by the manufacturer (Rotor-Gene Q, Venlo, The Netherlands).

The phylogenetic trees of VP1 and RdRp-encoding nucleotide sequences were inferred using IQ-TREE v1.6.12 [47 (link)] with 10,000 pseudo-replicates [48 (link)], incorporating the best-fit model of nucleotide substitution (VP1: TIM2 + F + R10, RdRp: GTR + F + R10) [49 (link)], and rooted by a midpoint. Trees were visualized with ggtree R-package [50 (link)].
Maximum clade credibility (MCC) trees for sequences with available collection dates were inferred for GI (N = 71 sequences) and GII (N = 915 sequences) using BEAST v.1.10.4 [51 (link)]. The best-fit partitioning scheme (GI: (1 + 2)(3), GII: (1,2,3)) and substitution models (GTR + I + G + X) for Bayesian analysis were chosen according to the Bayesian Information Criterion using the PartitionFinder 2 program [52 (link)]. For each genogroup, marginal likelihoods were calculated for combinations of coalescent tree priors (coalescent constant size, coalescent exponential growth) and molecular clock models (strict, relaxed log-normal) using the path sampling/stepping stone procedure implemented in BEAST v1.10.4 [53 (link)]. Then, different model settings were compared using the Bayes factor (BF) test. The combination of coalescent constant prior and relaxed lognormal molecular clock was strongly favored (log BF > 10) for both genogroups. The MCMC chains were run for 50 and 800 million steps with sampling every 5000 and 10,000 steps for GI и GII, respectively. The convergence of Markov chain Monte Carlo (MCMC) was inspected using Tracer v1.7 [54 (link)]. The maximum clade credibility (MCC) tree was annotated with TreeAnnotator v1.10.4 using 10% burn-in.

Complete genome sequences available for the genus Norovirus (n = 3439) were downloaded from the Genbank database as of July 2020. Sequences with more than 1% ambiguous nucleotides or more than five ambiguous nucleotides in a row were omitted from the dataset. The remaining ambiguous nucleotides were automatically resolved to a consensus using a custom Python script available at https://github.com/v-julia/resolve_ambiguous (accessed on 27 November 2022). The coordinates of ORF1, ORF2 and ORF3 were extracted from GenBank annotations. Then, the nucleotide sequences of ORFs were excised from the full genome sequences and aligned separately based on their corresponding amino acid translations using mafft v7.450 [30 (link)]. Next, the resulting nucleotide alignments of ORFs were concatenated, and the columns containing more than 20% gaps were removed using trimAl [31 (link)]. Since the ORFs were concatenated, their overlapping regions (17 nt between ORF1 and ORF2, 1 nt between ORF2 and ORF3) were duplicated in alignments. Finally, sequences sharing more than 99.5% identity were excluded. The resulting alignment of concatenated ORFs contained 1084 nucleotide sequences. The virus host and collection date were retrieved from GenBank entries automatically using custom python script and manually verified for all sequences in the dataset. The genogroups, genotypes, P-groups and P-types of viruses were designated using “Norovirus Typing Tool version 2.0” [32 (link)]. The final alignment, as well as scripts used for alignment preparation and data retrieval are available at https://github.com/orlovartem/NoV_recombination (accessed on 27 November 2022).