Analysis of GII.4 NoV strains of the novel variant GII.4 Sydney 2012 circulating in Italy in 2012-2013 identified recombinant strains possessing a polymerase gene derived from the former variant New Orleans 2009. These findings may pose a challenge for the diagnostic/characterization of recent GII.4 NoVs.

Noroviruses (NoVs) are a major cause of acute gastroenteritis in both children and adults. NoV can be classified genetically into at least six genogroups, GI to GVI (Green 2007; Martella et al., 2009). Although more than 30 genotypes within genogroup GI, GII and GIV may infect humans (Kroneman et al., 2013), a single genotype, GII.4, has been associated with the vast majority of NoV-related outbreaks and sporadic cases of gastroenteritis worldwide (Bok et al., 2009).

GII.4 NoV strains continuously undergo process of genetic/antigenic diversification and periodically generate novel strains via accumulation of punctate mutations or recombination, with novel GII.4 variants emerging every 2-3 years (Bull et al., 2010; SIebenga et al., 2007). Since 1996, distinct GII.4 variants have been associated with pandemics or major epidemics of NoV gastroenteritis, including US95/96 1996, Farmington Hills 2002, Asia 2003, Hunter 2004, Yerseke 2006a, Den Haag 2006b and New Orleans 2009 (Kroneman et al., 2013). In the late 2012, increased incidence of NoV-related outbreaks and/or illness in various countries has been related to the emergence of a novel GII.4 variant, Sydney 2012. This variant was first identified in March 2012 in Australia (van Beek et al., 2013) and it was found to have originated via recombination by acquiring a GII.Pe polymerase gene (ORF1) from a GII.4 variant Osaka 2007 strain and the ORF2 and ORF3 from a GII.4 Apeldoorn 2008-like strain (Eden et al., 2013).

The Italian Study Group for Enteric Viruses (ISGEV) monitors the epidemiology of enteric viruses in children through hospital-based surveillance. Monitoring and characterization of NoVs is achieved by a multi-target analysis in the diagnostic regions A, B, C and D of NoV genome (Kroneman et al., 2011) and interrogation of the Norovirus Typing Tool database.

During the late 2012 (November-December) and January 2013 ISGEV monitored a 28.9% prevalence (90/311) of NoV infection in children hospitalized or visited for gastroenteritis, vs a prevalence of 25.2% (77/305) in the same period (November-January) of the 2011-2012 winter season. In the 2012-2013 winter season, about 74.3% of the fully typed strains were characterized as GII.4 Sydney 2012, confirming that in Italy as in other European and extra-European countries this new NoV variant had become predominant (Giammanco et al., 2012).

For four samples inconsistencies were observed between region A (pol) and region C (cap)-based characterization, suggesting either mixed infections or a recombinant origin. These strains were found to have a GII.4 New Orleans 2009 pol and a GII.4 Sydney 2012 cap. The sequences of the four recombinant strains are available in GenBank under accession numbers KF378731 (PA13/2013/ITA), KF378732 (PR4200/2012/ITA), KF378733 (PA83/2012/ITA) and KF386146 (PR343/2013/ITA). The recombination event was mapped to the ORF1/ORF2 (pol/cap) junction region. This region is highly prone to recombination in NoVs (Bull et al., 2007), although other recombination hot spots have been identified in the ORF2/ORF3 overlap and in the junction of the shell and protruding capsid domains (Eden et al., 2013). In the four recombinant strains, the GII.Pe pol was replaced by a GII.P4 pol derived from the former pandemic NoV GII.4 variant New Orleans 2009. This variant was still dominant in Italy in the winter season 2011/2012 (41.7% of the detected NoV strains) and its circulation was documented until September 2012. Notably, one recombinant strain was detected as early as January 2012, almost contemporaneously to the emergence of the pandemic GII.4 variant Sydney 2012.

Also, we observed several distinctive punctate mutations in the GII.P4 New Orleans 2009 pol gene between the recombinant strains detected in Northern and Southern Italy, suggesting that independent recombination events occurred.

These findings may be relevant for the diagnostic, as these novel inter-pandemic recombinants can be identified only by multi-target analysis, i.e. by combined analysis of the pol and cap genes. Therefore, the GII.Pe pol, considered a signature of the pandemic NoV variant Sydney 2012, in some strains can be replaced. Continued surveillance for NoV infections and additional data on clinical and epidemiologic features will enable precise assessment of the public health implications of the new variant GII.4 Sydney 2012 and of its recombinant relative strains.

References

Bok K, Abente EJ, Realpe-Quintero M, Mitra T, Sosnovtsev SV, Kapikian AZ, Green KY. 2009. Evolutionary dynamics of GII.4 noroviruses over a 34-year period. J Virol 83:11890-11901.

Bull RA, Eden JS, Rawlinson WD, White PA. 2010. Rapid evolution of pandemic noroviruses of the GII.4 lineage. PLoS Pathog 6:e1000831.

Bull RA, Tanaka MM, White PA. 2007. Norovirus recombination. J Gen Virol 88:3347-3359.

Eden JS, Tanaka MM, Boni MF, Rawlinson WD, White PA. 2013. Recombination within the pandemic norovirus GII.4 lineage. J Virol 87:6270-6282.

Giammanco GM, De Grazia S, Tummolo F, Bonura F, Calderaro A, Buonavoglia A, Martella V, Medici MC. 2013. Norovirus GII.4/Sydney/2012 in Italy, winter 2012-2013. Emerg Infect Dis 19(8):1348-9.

Green KY. 2007. Caliciviridae, p. 949-979. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.

Kroneman A, Vega E, Vennema H, Vinjé J, White PA, Hansman G, Green K, Martella V, Katayama K, Koopmans M. 2013. Proposal for a unified norovirus nomenclature and genotyping. Arch Virol In press.

Kroneman A, Vennema H, Deforche K, v d Avoort H, Penaranda S, Oberste MS, Vinje J, Koopmans M. 2011. An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol 51:121-125.

Martella V, Decaro N, Lorusso E, Radogna A, Moschidou P, Amorisco F, Lucente MS, Desario C, Mari V, Elia G, Banyai K, Carmichael LE, Buonavoglia C. 2009. Genetic heterogeneity and recombination in canine noroviruses. J Virol 83:11391-11396.

Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, Siezen RJ, Koopmans M. 2007. Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol 81:9932-9941.

van Beek J, Ambert-Balay K, Botteldoorn N, Eden J, Fonager J, Hewitt J, Iritani N, Kroneman A, Vennema H, Vinje J, White P, Koopmans M. 2013. Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012. Euro Surveill 18:8-9.