Mutation in a Lordsdale norovirus epidemic strain as a potential indicator of transmission routes

Abstract

An increase in norovirus outbreaks was reported internationally during 2002 and 2003 and was also observed in Oxfordshire (United Kingdom) hospitals. To understand their epidemiological relationships, viruses from 22 outbreaks (15 from one hospital) were subjected to nucleotide sequencing. The 3'-terminal 3,255 nt or complete genomes were determined for 49 viruses. All outbreaks were caused by a genogroup II norovirus related to the Lordsdale virus (GII 4), common in healthcare settings. The norovirus mutation rate was sufficiently high that the 3,255-nucleotide sequences allowed separate and potentially connected outbreaks to be identified, since all outbreaks with identical sequences were temporally or geographically linked. The high mutation rate was further indicated by four mutations and three microheterogeneities in 3,255 nucleotides during 17 days of norovirus shedding by an immunocompromised patient. The data suggested that multiple virus introductions from the community, occasional transmission among wards, and one instance of ongoing environmental contamination had occurred. The accumulation, or lack, of mutations within an outbreak was also used to indicate the predominant transmission route. In an outbreak where person-to-person spread was thought to predominate, six mutations were detected throughout the genome, whereas one mutation was detected when point source infection was suspected. This norovirus epidemic strain differed from its closest previously described relative by 11.4 to 13.6% in the outer P2 domain of the capsid, which also had a single-amino-acid insertion. Alterations to the capsid structure compared to previous noroviruses may explain the increased number of outbreaks during 2002 and 2003.

FIG. 1.

Nucleotide sequences (285 nt) of the five closely related norovirus variants identified among 22 outbreaks (Table 1) and Lordsdale virus. The sequence is located within the RNA polymerase between primer pairs JV12Y and JV13I. Dots indicate sequence identity with variant 1. The 42 nt of primer sequence have been deleted from the 327-bp PCR amplicon. The locations of this sequence within the genome of strain Hu/NLV/Oxford/B5S22/2003/UK (GenBank accession no. AY581254) are indicated. The sequence equivalent to the 6-nt motif thought to indicate a new norovirus variant (16) is highlighted and boxed.

FIG. 2.

Neighbor-joining tree showing relationships among 49 strains from 18 outbreaks within the 3′-terminal 3,255 nt. The strains and outbreaks are indicated as follows: hospital (Hosp), plus ward where more than one outbreak from one hospital was included; date of sample collection (month/day/year); and sample number. *, variants shed over 17 days by an immunocompromised patient.

FIG. 3.

Relationships among capsid nucleotide sequences of eight of the Oxfordshire viruses, previous GII Lordsdale virus-like viruses, and other prototype noroviruses shown using a neighbor-joining tree. The input alignment file was generated using Clustal W version 1.8. The isolation years of the strains within the Lordsdale virus-like group are indicated with their corresponding clusters of variants. Bootstrap values are given at appropriate nodes. The GenBank accession numbers of the strains used are as follows: Dillingen/259/01/Germany, AF425766; Berlin/495/2000/Germany, AF427123; Koenigswusterhaus/120/2000/Germany, AF427121; 379/96019984/1996/Az, AF080556; 373/96019743/1996/SC, AF080555; Burwash Landing/331/1995/US, AF414425; 004/95 M-14/1995/AU, AF080551; Dijon171/1996/France, AF472623; Miami Beach/326/1995/US, AF414424; Berlin/238/98/Germany, AF425764; Beeskow/124/2000/Germany, AF427120; Oberschleissheim/112/1999/DE, AF427113; 416/97003156/1996/LA, AF 080559; Mora/1997/Sweden, AY081134; Parkroyal/1995/UK, AJ277613; Symgreen/1995/UK, AJ277619; Efurt/007/2000/Germany, AF427117; Hu/NV/Oxford/B5S22/2003/UK (a representative of the Oxfordshire viruses), AY581254; Bristol/1993/UK, X76716; UK3-17/12700/192/GB, AF414417; Lordsdale/1993/UK, X86557; Camberwell/1994/AU, AF145896; MD145-12/1987/US, AY032605; MD134-7/1987/US, AY030098; Toronto virus, U02030; Hawaii virus, U07611; Hillingdon virus, AJ277607; Snow Mountain virus, L23831; Melksham virus, X81879; Mexico virus, U22498; Desert Shield virus, U04469; Norwalk virus, M87661; and Southampton virus, L07418.

FIG. 4.

Alignment of the capsid amino acid sequences of the representative Oxfordshire variant Hu/NV/Oxford/B5S22/2003/UK (GenBank AY581254), its closest previously described relative (004/95 M-14/1995/AU [GenBank AF080551]), and Norwalk virus (GenBank M87661). The different subdomains of the capsids, estimated using the previously described crystal structure of the Norwalk virus capsid (26), are indicated. The majority of amino acid sequence changes between the new strain and its previous closest relative occurred in the outer P2 subdomain of the capsid and are highlighted in boxes.