Noroviruses (NoV) are the most common cause of acute viral gastroenteritis 
, with the numbers of reported outbreaks peaking characteristically between November and March in the northern hemisphere 
. Illness is usually self-limiting and symptoms, comprising acute onset vomiting and watery diarrhea, subside within one to three days 
. The relevance of studying NoV lies in their high prevalence in the population 
, and in the more severe and prolonged illness that is seen among elderly and immunosuppressed patients 
. NoVs are highly infectious, due to the combination of an extremely low infectious dose (an estimated ID50 of less than 20 viral particles 
), very high levels of shedding (around 108
but up to >1010
RNA copies per gram of stool) and prolonged shedding after clinical recovery 
. NoV outbreaks, which may affect hundreds of people and are notoriously difficult to control, are primarily associated with places where people are in close contact, for example hospitals and long-term care facilities.
NoVs are a genetically diverse group of positive sense single-stranded RNA viruses from the Caliciviridae family. Their 7.5 kb genome includes three open reading frames (ORFs). The first ORF encodes a polyprotein that is post-translationally processed to form the non-structural proteins, the second and third ORFs encode the major and minor structural proteins; VP1 or the capsid protein and VP2. The viral capsid is formed by 180 copies of the major capsid protein, and governs antigenicity, host-specificity and environmental stability.
NoVs are classified into five distinct genogroups, which are further subdivided into genotypes, based on their amino acid capsid sequence. Molecular epidemiological studies have shown that in recent years approximately 70% of NoV outbreaks among humans have been caused by one dominant genotype, GII.4 
With continuous surveillance systems in place in some countries since the mid-1990s, it has become apparent that the number of reported NoV outbreaks, and especially those caused by GII.4 strains has risen since the appearance of the 2002 variant of GII.4 
. Since then, genetically distinct GII.4 variants have emerged, and spread rapidly across the world causing epidemic waves of NoV illness 
. To date, three variants named after the year when they were first detected have been identified in populations across the world (the 2002, 2004 and 2006b variants 
). The emergence of each of these three variants was followed by ‘hot’ NoV winters with sharply increased numbers of reported outbreaks. Older strains belonging to the lineage designated 1996 were also detected around the world, although surveillance was limited at that time.
The pattern of continuous lineage turnover, referred to as epochal evolution 
, with emerging new variants replacing previously predominant circulating ones, is strongly reminiscent of what is observed in the molecular epidemiology of Influenza A virus (IAV). The evolutionary interaction between IAV and the human immune response results in antigenic drift, illustrated by the characteristic ladder-like tree shapes for hemagglutinin and neuraminidase surface proteins 
. Long-term partial immunity to the virus induces sharp fitness differences among strains and drives rapid amino acid replacement at key antigenic sites, pinpointed by in vitro
and in silico
. Whereas antigenic data can be readily generated for IAV, allowing the comparative mapping of antigenic and genetic evolution 
, research of NoV antigenic properties has been hampered by the lack of a simple cell culture model 
. However, recent publications indicate that the genetic differences between NoV genotypes, and also between variants of the GII.4 genotype, translate into distinct antigenic types, although molecular determinants remain largely unclear 
. Thus, individuals may be repeatedly infected by strains belonging to different genotypes, and also, because immunity against NoV infection is short-lived at best 
, possibly repeatedly by strains of the same genotype. As a result, the impact of immune responses on NoV epidemiology remains poorly understood and phylodynamic and molecular adaptation studies may provide some key insights.
In this study, we aimed to provide a rigorous measurement of NoV GII.4 diversity through time, and we investigated viral population expansions in relationship to the increased numbers of infections reported in recent years. Evolutionary and population dynamics of GII.4 NoVs were estimated by a Bayesian coalescent approach, using two different datasets of sequences from strains with known detection dates, between 1987 and 2008. One set of sequences contained full capsid sequences, the other short partial polymerase sequences, which had been obtained for standard-procedure genotyping in NoV surveillance in Europe 
) and from the global NoV surveillance network Noronet 
We also tested whether these dynamics differed from neutral expectations, so whether and how they were shaped by selective pressure, and we attempted to further elucidate the molecular determinants of NoV evolutionary and epidemiological dynamics using in silico techniques. To identify the molecular characteristics of NoV GII.4 strain replacement, we investigated both directional and diversifying selection and elucidated capsid protein positions showing evidence for co-evolutionary dynamics acting between sites.