We have used influenza virus whole-genome sequences combined with sophisticated evolutionary analysis to estimate the molecular epidemiological characteristics of pandemic H1N1 influenza in the United Kingdom. The distribution of United Kingdom-specific clusters and individual United Kingdom isolates throughout the global pandemic H1N1/09 virus phylogeny proves that the epidemic in the United Kingdom resulted from multiple independent introductions of the virus into the country during the first wave of infection. None of the viruses analyzed in this study, more broadly as part of United Kingdom surveillance, or worldwide showed any evidence of significant antigenic divergence from A/California/07/2009, although they are genetically distinct.
Epidemiological tracking of cases during this period suggested that there were laboratory-confirmed cases from at least 60 distinct introductions, grouping into at least 9 different epidemiologically defined clusters. In addition to the 13 United Kingdom clusters we defined here (containing 94 of the 153 isolates sequenced), there were 52 isolates, widespread geographically in the United Kingdom, distributed throughout our phylogeny. This suggests that our sampling of the lineages that arrived in the United Kingdom at the start of the outbreak was reasonably complete and that the 13 clusters and 52 dispersed lineages account for much of the United Kingdom pandemic H1N1/09 virus genetic diversity. Standard power calculations to add support to this observation are not possible, however, as sequences are not statistically independent observations but are in fact highly correlated due to the presence of shared ancestry.
Based on our analysis, at least one United Kingdom cluster (cluster UKA2-GC3) began to diverge 1 week (95% HPD range, 1 to 14 days) before the first clinically diagnosed United Kingdom case. The node which connects clusters UKA1-GC3 and UKA2-GC3, although poorly supported (posterior probability = 0.3542), has a time of most recent common ancestor (TMRCA) of 13 April 2009, suggesting that pandemic H1N1/09 virus was already spreading undetected in the United Kingdom around the same time that it was identified in Mexico, presumably as a result of unidentified infections in returning travelers. Note that there were approximately 5,000 travelers per week to and from Mexico in the weeks immediately preceding the first confirmed introduction to the United Kingdom.
The reasons for the apparent lag between the introductions of pandemic H1N1/09 virus into the United Kingdom and the peak of first-wave infections are not known. The spread of A/H1N1/09 in mainland Europe during the first wave in the United Kingdom was characterized by sporadic cases and isolated self-limiting outbreaks linked to importations. In the United Kingdom, however, the major generalized epidemic occurred in June and July and declined only once schools closed for the summer holidays at the end of July (6
). No equivalent epidemic wave was reported in Europe until the autumn, during which time the United Kingdom had its second wave of infection. The beginning of the second wave in the United Kingdom also coincided with the reopening of schools after the summer holiday period. It is possible that the United Kingdom public health response, which was comprehensive and applied consistently during the early phases and which consisted of laboratory confirmation of suspected cases followed by antiviral prophylaxis of contacts, may have contributed to this delay. Antiviral prophylaxis appears to be effective at reducing household transmission (R. G. Pebody et al., unpublished data), suggesting that the strict prophylaxis policy acted to extinguish some of the early viral introductions, possibly slowing the rate of rise of the pandemic.
We were able to show clear evidence of virus lineage persistence in the United Kingdom between the first and second epidemic waves. A prominent characteristic of influenza epidemics in temperate regions is their seasonality, with the majority of infections occurring during the winter months in the northern and southern hemispheres, in contrast to year-round influenza virus circulation across tropical regions. This has led to the hypothesis that influenza virus genetic diversity is generated continually in tropical regions, primarily Southeast Asia, from where viral lineages migrate to temperate regions each year, founding the next seasonal epidemic (18
). Here we found two United Kingdom-specific clusters that appeared in the first wave of infection and persisted into the second wave of infections. The density of our sampling and phylogenetic inference suggest that this reflects true persistence rather than reintroduction within the United Kingdom between the epidemic waves. Additional HA gene sequencing of influenza virus isolates between the first and second waves showed that viruses of cluster UKK-GC7, which arose in the United Kingdom in June and possessed the signature amino acid substitution D222E, were present and circulating in the United Kingdom between July and September 2009. The likelihood of persistence is further supported by intensive surveillance as a consequence of the national response to the pandemic, which clearly indicates that there were low levels of infection sustained in the community in individuals without any history of travel abroad, as well as sporadic travel-associated cases. In 2009, persistence may have been facilitated by the timing of the waves, as the majority of first-wave infections occurred during the summer months in the United Kingdom (with a peak in late July), whereas the second wave of infections started in September and peaked earlier (October-November) than usual for seasonal influenza epidemics. Consequently, the time between waves was much shorter than that for typical seasonal influenza, where the length of time between the end of one winter wave and the onset of an autumn wave may be as much as 8 to 9 months. Furthermore, a recent study also suggested that the occurrence of two consecutive waves in the United Kingdom, as opposed to only sporadic cases in the rest of Europe during the summer, was a consequence of a relatively large number of early importations (as demonstrated here) combined with a low level of absolute humidity, which has been shown to affect both transmissibility and the survival of influenza virus (20
). The relative immunological naïvety of the human population to the pandemic H1N1/09 variant may also have enabled higher-than-usual infection rates during the interwave period. Whatever the underlying mechanism, it seems that influenza virus continued to circulate in the United Kingdom between infection peaks without causing a sustained level of clinical disease.
It is important to consider the benefits that are brought by real-time monitoring of virus genome diversity during a pandemic or epidemic. The very-high-resolution tracking of individual lineages is achievable only with full genome sequences. Coupled with spatial information, we show that detailed insight into transmission chains and epidemiology becomes apparent, extending what can be observed by use of sequences alone (1