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. 2018 Mar 2:9:371.
doi: 10.3389/fmicb.2018.00371. eCollection 2018.

Intrahost Norovirus Evolution in Chronic Infection Over 5 Years of Shedding in a Kidney Transplant Recipient

Andrej Steyer  1 , Tilen Konte  2 , Martin Sagadin  1 , Marko Kolenc  1 , Andrej Škoberne  3 , Julija Germ  1 , Tadeja Dovč-Drnovšek  4 , Miha Arnol  3 , Mateja Poljšak-Prijatelj  1
Affiliations

Intrahost Norovirus Evolution in Chronic Infection Over 5 Years of Shedding in a Kidney Transplant Recipient

Andrej Steyer et al. Front Microbiol. .

Abstract

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope "hot-spots" of modified and optimized norovirus-host interactions.

Keywords: HBGA binding; chronic infection; molecular evolution; norovirus; solid organ transplantation.

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Figures

Figure 1
Figure 1
Single nucleotide polymorphism accumulation in the NV RdRp gene region, VP1 gene region, and P2 domain of the VP1 gene region over time (see also Table 1).
Figure 2
Figure 2
Schematic representation for specific samples (as indicated) for the variable sites (single nucleotide polymorphism occurrence and variant frequency) throughout the amplicon that contained the NV RdRp gene and VP1 gene regions.
Figure 3
Figure 3
Maximum likelihood phylogenetic tree for the haplotypes generated for the VP1 gene region along with the most identical GII.4 Den_Haag 2006 variant strains from GenBank and other variants for comparison. Haplotype designation is as given in Table 1, as the patient code (p1), the sampling point (t1-t9), and the frequency of the generated sequence (f0.08–f1.00).
Figure 4
Figure 4
Analysis of the partial P-domain alignment of the haplotypes (residues 289–445). Commonly defined epitopes and HBGA binding sites are highlighted as the colored columns. Green, epitope A; yellow, epitope B; orange, epitope C; pink and violet, epitope D; cyan, epitope E; blue and violet, HBGA binding sites. The sites within the 8-Å expanded area of the epitopes are indicated with letters above the alignment, colored as for the epitopes. The Jalview histogram below the alignment indicates the conservation of the physico-chemical properties for each column (lower bars with lower numbers, lower conservation; completely conserved columns are in gray). The positive and negative selection pressures defined through the Datamonkey algorithms are shown as red plus and black minus signs, respectively.
Figure 5
Figure 5
Surface model representations of the P domain. The epitopes and HBGA binding pockets are colored as in Figure 4, and are superimposed over the red sites defined with positive diversifying selection pressure. Black numbers, sites in the chain A monomer (light gray); black numbers with apostrophe, sites in the chain B monomer (dark gray); green, epitope A; yellow, epitope B; orange, epitope C; pink and violet, epitope D; cyan, epitope E; blue and violet, HBGA binding sites. Left, top view; center, side view; right, view of the chain A/B interface.
Figure 6
Figure 6
Molecular modeling of the interactions of VP1 site 393 with Leb HBGA. Superposition of the 4OPO crystal structure of strain Saga4 in complex with HBGA type Leb tetraglycan (yellow sticks), and the models of p1t1_f1.00 (violet sticks), p1t7_f0.69 (orange sticks), and p1t9_f0.25 (green sticks). Ser, serine; Asn, asparagine; Lys, lysine; FUC, fucose; LeFUC, Lewis fucose; GlcNAc, N-acetylglucosamine; GAL, galactose. Dashed lines show distances from side chain atoms of residues at site 393 to LeFUC and GlcNAc.
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