Genetic analyses of WG sequences showed that HAV variants identified from food-borne outbreaks are heterogeneous, though closely related, despite sharing the VP1/P2B junction sequences. The degree of genetic diversity among HAV variants from a single outbreak varied, with HAV WG sequences being different at up to 23 nt sites among the onion and oyster-related outbreaks. The HAV sequence variants identified in each outbreak belonged to more than one strain. Although only one dominant strain was found in outbreak A, two to three dominant strains were identified in the other three outbreaks associated with consumption of green onions or oysters. Additionally, each outbreak was associated with many minority strains ().
The origin of such diversity among HAV variants in each outbreak is not known. It is conceivable that the observed genetic heterogeneity reflects diversity of viral strains circulating at the geographic location of the source of the implicated food items rather than the generation of viral variants from a single HAV ancestor during the course of infection of each host. The existence of more than one dominant strain in outbreaks B, C and D supports heterogeneous HAV sources rather than acquisition of identical substitutions by a single source-strain in groups of infected patient. However, it is possible that one of the dominant variant in each outbreak represents the ancestral strain; then, the other dominant variants in outbreaks B, C and D could have originated from the ancestral strain through extensive parallel evolution in several infected patients. Although, such frequent, independent acquisition of identical substitutions seems unlikely even under strong selection pressures. Alternatively, the observed genetic diversity could have been developed through a chain of transmissions. It is noteworthy that during outbreak C the contaminated onions were consumed in a single restaurant over a few days 
, which argues against the existence of widespread chains of transmission. Therefore, close genetic relatedness among HAV strains found in each outbreak are more likely to have resulted from viral evolution among many hosts in the locale where the contaminated food originated.
Consumption of food contaminated with a diverse HAV population may result in co-infection of each individual with more than one HAV strain. Detection of the intra-host HAV diversity reported earlier 
seemingly supports this suggestion. However, genetic analysis of intra-host HAV variants from individuals involved in the onion-related outbreak B showed a lack of detectable intra-host genetic diversity. Food items associated with the outbreaks studied here are usually washed during preparation, thus diluting the viral pool. Although HAV is tolerant to adverse environmental conditions for extended periods of time 
, it is approximately100-fold less infectious via peroral than intravenous inoculation as was shown in experimental infections of nonhuman primates 
. Thus, the observed intra-host homogeneity can be explained by exposure of each individual to the limited number of viral particles, from which a single variant or few variants successfully established productive infection. The existence of HAV intra-host heterogeneity was not studied extensively in this study. However, recent publications have reported intra-host HAV heterogeneity in infected patients 
in contrast with our findings. Although this discrepancy can be attributed to differences in the employed experimental methodology for the detection of intra-host heterogeneity, it could also indicate variation in the number of HAV variants establishing infection in exposed individuals. Exposure to large inocula of HAV, from raw sewage for example, can be expected to lead to productive infection by a heterogeneous HAV population. By contrast, exposure to a small number of viral particles, as from contaminated food, would more likely result in productive infection by a homogeneous population. Thus, analysis of intra-host heterogeneity may provide important clues for characterization of the potential modes of HAV transmission. Further molecular investigations of HAV variants from outbreaks associated with different viral sources are warranted to uncover the association between the intra-host HAV diversity and modes of transmission.
Analysis of WG sequences offers a more complete genetic characterization of HAV strains than short subgenomic regions. In this study, HAV genetic identity was more precisely determined using WG sequences. Ten of 14 sporadic HAV strains that shared the VP1/P2B region with outbreak strains were clearly separated from these strains (). However, 4 sporadic strains were genetically close to strains from 2 outbreaks, suggesting their link to the outbreaks despite the lack of a clear epidemiological association between these sporadic and outbreak strains.
The molecular analysis showed that none of the 500 nt-regions accurately reflect genetic relatedness among HAV strains, suggesting a cautious use of subgenomic regions for the molecular tracking of transmissions. However, it should be noted that tracking does not require assessment of complete phylogenetic relationships and is plainly based on the identification of genetic proximity or identity. Additionally, the high prevalence of infection with genetically related HAV variants during the epidemiologically identified outbreaks significantly reduces the opportunity for false detection of transmissions, thus allowing for the reasonably efficient use of subgenomic regions. Identification of the sporadic and outbreak HAV strains sharing the VP1/P2B region indicates that tracking transmissions using subgenomic regions may be misleading when applied to HAV strains with unknown epidemiological association. These findings indicate that investigations should be conducted using HAV WG sequences.