Novel GII.12 Norovirus Strain, United States, 2009–2010

In October 2009, a novel GII.12 norovirus strain emerged in the United States and caused 16% of all reported norovirus outbreaks during the winter season. Sequence analysis demonstrated a recombinant virus with a P2 region that was largely conserved compared with previously sequenced GII.12 strains.


Everardo Vega and Jan Vinjé
In October 2009, a novel GII.12 norovirus strain emerged in the United States and caused 16% of all reported norovirus outbreaks during the winter season. Sequence analysis demonstrated a recombinant virus with a P2 region that was largely conserved compared with previously sequenced GII.12 strains.
N oroviruses are the leading cause of viral gastroenteritis outbreaks in the United States (1). Over the past decade most norovirus outbreaks have been caused by genogroup (G) II.4 noroviruses, while each of the other genotypes did not cause >7% of the outbreaks (2,3). However, previous studies have suggested that non-GII. 4 noroviruses have been predominant in the past (3,4). For example, analysis of archived samples from 1974 through 1991 has shown that the frequency of GII.3 was 48% compared with 16% for GII.4 and 14% for GII.7 strains (3). Therefore, it is essential to study sudden increases of non-GII.4 strains to determine possible signatures that could be associated with increased transmissibility or population susceptibility. In this article, we describe the emergence of a novel GII.12 strain in the United States in the winter of 2009-10 that was associated with a large number of the norovirus outbreaks.

The Study
From October 2009 through June 2010, fecal specimens from patients affected by 194 outbreaks from 21 states were submitted to the Centers for Disease Control and Prevention (Atlanta, GA, USA); 39 (20%) of the viruses were typed as GII.12 by phylogenetic analysis by using region D sequences (5). During the same period, CaliciNet data confi rmed an identical GII.12 strain that caused 67 (14%) of the 469 outbreaks reported by 12 states (6). To further study these new strains, we amplifi ed the P2 region from 38 GII.12 outbreaks and 3 GII.    (7) and Hungary in 2009 (Figure 1).
Phylogenetic analysis of P2 sequences from all GII.12 strains indicated a temporal pattern, with the new strains clustering separately from GII.12 strains detected before 2009 (online Appendix Figure). The new GII.12 strains clustered with GII.12 strains from Australia and Hungary in both the P2 and region A (5) (Figure 1). A single amino acid change in P2, at aa 392, occurred consistently in the new strains compared with archival GII.12 noroviruses (Figure 2). Additional amino acid substitutions were identifi ed outside the P2 region at positions 22, 47, and 465 ( Figure 2). Partial RNA-dependant RNA polymerase sequences confi rmed that the new GII.12 strains were recombinant viruses, as reported previously (7) ( Figure  1). Because different norovirus polymerases may have different nucleotide incorporation rates (8), and thus could play a role in enhanced replication effi ciency of the new GII.12 strains, we amplifi ed and analyzed a partial region of the polymerase gene but found no differences between the GII.12 strains pre-or post-2009.

Conclusions
A novel GII.12 norovirus strain emerged in winter 2009-10 and caused 16% of the norovirus outbreaks in the United States. Sequence comparison with archival GII.12 strains demonstrated that even though there were clear and distinct nucleotide changes in the P2 region, there were few amino acid changes in the complete viral protein (VP) 1 ( Figure 2). The increase in the number of GII.12 outbreaks (online Appendix Figure) was confi rmed by data from CaliciNet, which indicates that the increase was not caused by surveillance bias (6). Both P2 as well as polymerase sequence analysis demonstrated the emergence of a recombinant strain without novel amino acid substitutions in the P2 region, as has been reported for emerging GII.4 variants (9). All nucleotide substitutions throughout the P2 region of the new GII.12 strains, compared with pre-2009 GII.12 strains, were synonymous or unstable mutations ( Figure 2).
Most norovirus evolution studies have focused on the VP1, more specifi cally the P2 region, where the antigenic and histo-blood group antigen attachment sites are located (7,9,10). The novel GII.12 virus had 1 unique amino acid substitution in the P2 domain but with similar biochemical properties, suggesting that the emergence of this virus may have been caused by virulence features coded by signatures outside of the P2 region or by changes in population susceptibility. In contract to a recent report (11), our results suggest that the nonstructural proteins or regions outside of the P2 region may play a role in the evolution and virulence of norovirus strains. Substitutions in some key amino acids in the N-terminal region of the capsid have been speculated to be involved in the formation of secondary structures for effi cient initiation of translation of VP1 (12).
The fact that 16% of all reported outbreaks were caused by a rare genotype highlights the importance of norovirus strain typing, which may provide insights into identifying which viral and/or host factors enable the emergence of novel norovirus strains. CaliciNet, which demonstrated its usefulness in this study, will be an important tool for monitoring changing trends and emergence of novel strains. However, until a cell culture system, small animal model, or infectious clone for human norovirus is available, the role of structural or nonstructural genes on norovirus pathogenicity and transmissibility will be diffi cult to assess.