Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence

Abstract

Viruses within the genus Norovirus of the family Caliciviridae are the major cause of acute, nonbacterial gastroenteritis worldwide. Human noroviruses are genetically diverse, with up to 57% divergence in capsid protein sequences, and comprise three genogroups. The significance of such genetic diversity is not yet understood. The discovery of murine norovirus (MNV) and its ability to productively infect cultured murine macrophages and dendritic cells has provided an opportunity to determine the functional consequences of norovirus diversity in vitro and in vivo. Therefore, we compared the full-length genomes of 21 new MNV isolates with five previously sequenced MNV genomes and demonstrated a conserved genomic organization consisting of four open reading frames (ORFs) and a previously unknown region of nucleotide conservation in ORF2. A phylogenetic analysis of all 26 MNV genomes revealed 15 distinct MNV strains, with up to 13% divergence at the nucleotide level, that comprise a single genotype and genogroup. Evidence for recombination within ORF2 in several MNV genomes was detected by multiple methods. Serological analyses comparing neutralizing antibody responses between highly divergent strains suggested that the MNV genogroup comprises a single serotype. Within this single genogroup, MNV strains exhibited considerable biological diversity in their ability to grow in culture and to infect and/or persist in wild-type mice. The isolation and characterization of multiple MNV strains illustrate how genetic analysis may underestimate the biological diversity of noroviruses and provide a molecular map for future studies of MNV biology.

FIG. 1.

The virulence of plaque-purified MNV1 clones differed in vivo. (A) MNV1.CW3, passage 3 (P3), and MNV1.CW8, passage 3, were virulent in STAT1^−/− mice. STAT1^−/− mice were orally inoculated with 3 × 10⁴ PFU of virus. The numbers of mice analyzed are indicated in parentheses. Differences in survival rates corresponding to MNV1.CW1, passage 3, and MNV1.CW3, passage 3 (P = 0.0109), and to MNV1.CW1, passage 3, and MNV1.CW8, passage 3 (P = 0.0017), were analyzed by the log rank test. (B) Plaque-purified MNV1 clones showed similar growth rates in vitro. RAW cells were inoculated at a multiplicity of infection of 0.05. Error bars represent the standard error of the mean of results from two independent experiments. No differences between the growth curves were detected by analysis of variance (P = 0.9983).

FIG. 2.

Alignment of multiple full-length MNV genomes. (A) The genome organization was conserved among all sequenced MNV genomes. The presence of a viral protein (VpG) linked to the genomic (G) and subgenomic (SG) RNA is predicted, but not proven, for MNV. The nucleotide positions of insertions and deletions identified in specific MNVs are indicated, as is the beginning of a 47-nucleotide (nt) region of nucleotide conservation in ORF2. Nucleotide numbering is based on the MNV1 sequence (GenBank accession number AY228235). Insertions are underlined, while deletions are indicated in lowercase. A truncated ORF3 coding region caused by the insertion in CR18 is also shown. The P domain of VP1 is shaded in gray. (B) The 5′ ends and ORF1-ORF2 junctions of all sequenced MNV genomes were conserved. Thirteen nucleotides conserved at the 5′ ends of the genomic and subgenomic RNAs are shaded in gray. Inverted repeats that form the stems of hairpins predicted using mfold (version 3.2) (51, 83) are represented by open arrows. (C) The 3′ UTRs of all sequenced MNV genomes had conserved and variable regions. Conserved nucleotides are shaded in gray. Inverted repeats that form the stems of hairpins predicted using mfold (version 3.2) (51, 83) are represented by open arrows.

FIG. 3.

Phylogenetic analysis of MNVs. (A) All sequenced MNV genomes comprise a single genogroup (GG). A consensus Bayesian tree based on VP1 sequences from MNVs and from prototype strains of other norovirus genogroups is shown. Posterior probabilities are presented next to the branches (1.00 is equivalent to 100% of the trees found in 1,000,000 MCMC generations contained in that particular grouping). Estimated sequence divergence, 0.1, or 10%. Sequence designations were assigned as follows: name-year, country code. Country codes: DE, Germany; NLD, The Netherlands. (B) All sequenced MNV genomes comprise a single genotype. The distribution of pairwise sequence divergences for comparisons of VP1 sequences from MNVs and from prototype strains of other norovirus genogroups is shown. Uncorrected pairwise sequence divergences were calculated using PAUP* (72). (C) All the sequenced MNV genomes comprise 15 distinct strains. A consensus Bayesian tree based on full-length MNV genomes is shown. Genetically distinct MNV strains are circled. For clarity, the names of plaque-purified MNV1 clones have been removed from the tree and are represented by branches.

FIG. 4.

Detection of recombinant MNVs. (A) Consensus Bayesian trees constructed using the whole genome, ORF1, and ORF2 nucleotide sequences are shown. MNVs with discordant positions are circled. A posterior probability of 0.95 or greater is represented by an asterisk. (B) The nucleotide similarity between the putative recombinant viruses CR7 and CR15 and the two nonrecombinant viruses CR1 and CR18 is shown. Nucleotide similarity was plotted using Simplot2 (http://sray.med.som.jhmi.edu/RaySoft/simplot_old/Version2/SimPlot_Doc_v24.html). A window size of 200 nucleotides with an increment of 20 was used. For similarity, 1.0 equals 100%. The P domain coding region of ORF2 is indicated by gray shading.

FIG. 5.

Many new MNVs exhibited altered plaque morphology. RAW cell monolayers were infected with dilutions of WU11, WU26, CR3, or MNV1.CW3, passage 4, and processed by plaque assay. Plaques were visualized with neutral red staining or fixed with acid alcohol, incubated with a 1:5,000 dilution of rabbit polyclonal serum raised against MNV1 (78), and visualized using the rabbit immunoglobulin Vectastain Elite ABC reagent and a 3,3′-diaminobenzidine kit (Vector Laboratories, Inc., Burlingame, CA). Pictures of wells with dilutions showing individual plaques were taken.

FIG. 6.

Serological analysis suggested that MNVs comprise a single serotype. (A) Sera raised against other MNV strains reacted with MNV1.CW3. Antisera were raised against WU11, CR1, CR3, CR6, CR7, and MNV1.CW3, passage 4. A serial dilution of each antiserum was incubated with purified virions of MNV1.CW3, passage 5, and reactivity was measured by an ELISA. Significant differences in the reactivity of the anti-MNV1.CW3 serum (P < 0.03) and the anti-CR7 serum (P < 0.04) were detected by the paired t test. Error bars represent the standard error of the mean of results for three independently generated antisera for each virus. OD₄₀₅, optical density at 405 nm. (B) Sera raised against other MNV strains neutralized MNV1.CW3. A serial dilution of each antiserum was incubated with 3 × 10⁴ PFU of MNV1.CW3, passage 4. The limit of detection of the plaque neutralization assay is represented by a dashed line. No significant differences in neutralization were detected by analysis of variance (P = 0.9465). Error bars represent the standard error of the mean of results for three independently generated antisera for each virus.

FIG. 7.

MNV1.CW3 was rapidly cleared from wild-type mice. C57BL/6 mice were orally inoculated with 3 × 10⁷ PFU of MNV1.CW3, passage 5. Tissue samples were harvested at 3 (D3), 5 (D5), and 7 (D7) days after inoculation. Virus titers were measured by plaque assays. Significant differences in mean titers were detected by the Mann-Whitney test. DI, distal ileum.

FIG. 8.

Several new MNV strains persistently infected wild-type mice. C57BL/6 mice were orally inoculated with 300 TCID₅₀s of WU11, CR1, CR3, CR6, CR7, or MNV1.CW3, passage 4. Tissue samples were harvested at 3 (D3), 7 (D7), and 35 (D35) days after inoculation. (A) Virus titers in the MLN and distal ilea (DI) were measured by the TCID₅₀ assay. The limit of detection of the TCID₅₀ assay is represented by a dashed line. Significant differences in mean titers compared to those of MNV1.CW3, passage 4, were detected using the Mann-Whitney test. (B) The number of genome copies in a single fecal pellet was measured using qPCR. The limit of detection of qPCR is represented by a dashed line. Significant differences in mean numbers of genome copies compared to those of MNV1.CW3, passage 4, were detected using the Mann-Whitney test. (C) The numbers of genome copies in various tissues at 35 days after inoculation were measured using qPCR. (D) The seroconversion of mice inoculated with MNV was measured by an ELISA. The background optical density at 405 nm (OD₄₀₅) is represented by a dashed line. Significant differences in mean OD₄₀₅ compared to that of MNV1.CW3, passage 4, were detected using the Mann-Whitney test.