Rotaviruses and noroviruses are important causative agents of childhood diarrhea, particularly in developing countries 
. Following the discovery of rotaviruses in 1973 
and noroviruses in 1972 
, the LID collaborated with the WHO to assess the role of viruses in childhood diarrhea 
. With consistent evidence that rotaviruses were a leading cause of diarrhea worldwide, the WHO recommended rotavirus vaccine initiatives as a global health priority 
. Norwalk virus was not detected in infants and young children in these early studies, although our current study supports an emerging consensus that noroviruses are second in importance to rotaviruses as agents of severe pediatric gastroenteritis 
Our analyses of these archival fecal specimens revealed that both rotaviruses and noroviruses circulated throughout the developing world in the late 1970’s. Rotaviruses were generally more prevalent than noroviruses, with the exception of Cayenne, French Guiana. The most common circulating rotavirus and norovirus genotypes were consistent with those generally observed to be most prevalent today (i.e. GII noroviruses and G1 rotaviruses) 
, and co-infections with representatives of both viruses were observed in multiple regions.
Certain rotavirus strains detected in these archival specimens are of particular interest as they represent unusual VP7 genotypes or VP7/VP4 genotype combinations. One is a G2P
rotavirus detected in a child in Entebbe, Uganda in 1976. The first G2P
genotypic combination (strain 1076) was isolated from a neonate in Sweden with an asymptomatic rotavirus infection in 1975 
. Our identification of this combination in Entebbe indicates that both asymptomatic and symptomatic rotavirus infections were associated with the G2P
genotypic combination that circulated in different parts of the world in that time period. Moreover, in a review of studies published during 1997–2006, the G2P
genotype was detected in 10% of African children with acute gastroenteritis, indicating that this strain has become established in more recent years in Africa 
In addition, G5 and G9 rotavirus strains were detected in a 4.5 month old and a 5 month old infant, respectively, in Hong Kong, China in 1978. G9 rotaviruses have been emerging in prevalence globally since the mid-1990’s 
, but the detection of Hu/RV/HK75/China/1978 indicates that this genotype has been circulating since at least 1978 in Asia. The VP7 gene of strain HK75 clustered most closely with those of the oldest lineage (I) of G9 rotaviruses, which no longer are known to circulate today, as they were replaced over time by phylogenetic lineages comprised of contemporary strains (II and III) 
. HK75 is also the oldest known Lineage I strain and the immediate precursor to all contemporary lineages. It is of interest that Lineage I G9 strains have been shown to exhibit the broadest neutralizing cross-reactivity, as they neutralize Lineage II and III G9 strains to high titer, making them ideal vaccine candidates 
. Previous analyses of G5 rotaviruses suggested that zoonotic or natural reassortment events may have given rise to G5 diversity 
. Our analysis confirmed the presence of three previously defined G5 lineages 
, each associated with at least one zoonotic event. All human strains clustered within two lineages (I and III), and both lineages also contained porcine strains, but no other types of animal strains, suggesting that swine may be involved with zoonotic transmission to humans. Lineage II contained no human strains, but closely related animal strains (swine, cattle, and horses). Strain HK69 predates the oldest known human G5 strains from Brazil 
, which indicates that any natural reassortment or zoonotic event may have occurred as early as 1978.
This study identified a variety of norovirus genotypes, and an evolutionary analysis of full-length norovirus VP1 genes illustrated this diversity. Norovirus genotypes from this cohort were diverse globally, indicating no evident trend in distribution. A previous evolutionary analysis of GII.3 noroviruses suggested that, although these viruses accumulate mutations over time, they tend to eventually revert back to the amino acid composition of older strains 
. Our analysis supports this finding and suggests that other norovirus genotypes may exhibit similar characteristics (GI.3, GI.5, GI.6, GII.6, GII.7, and GII.17).
Genotype GII.4 noroviruses are the most common strains associated with outbreaks of norovirus gastroenteritis worldwide 
. However, GII.4 noroviruses detected in this study (n
6) were outnumbered by GII.2 (n
10) and GI.3 (n
8) noroviruses, an observation that could reflect sampling procedures or the distribution of genotypes at that time. The original objective of this study was to document cases of viral associated diarrhea in specific settings globally, and not necessarily to characterize outbreaks. One of the six GII.4 norovirus strains detected in this collection (Hu/NoV/C127/FrenchGuiana/1978/GII.4) had sufficient RNA quality for full-length VP1 capsid sequencing and phylogenetic analysis; this strain clustered with the oldest GII.4 variants from Children’s Hospital, Washington DC (1974–1977) 
. However, two additional genogroup GII variants with closest homology to the GII.4 genotype (Hu/NoV/KL45/Malaysia/1978/GII.na and Hu/NoV/T091/Tunisia/1976/GII.na) were also detected in specimens collected in a similar time frame, but did not meet the criterion for belonging to GII.4 (>14.3% aa distance) 
. Furthermore, strains KL45 and T091 were of sufficient sequence dissimilarity for each to be proposed as a new genotype (>14.3% aa distance) 
. These GII variants have no contemporary counterparts, since none were available in GenBank, despite the ability to detect any closely homologous variants using the standard, widely used norovirus diagnostic primers employed in this study. Thus, strains KL45 and T091 came from unknown sources and caused single episodes of norovirus gastroenteritis without being part of the diverse repertoire of epidemiologically relevant noroviruses that still circulate today. Each of these viruses was detected in stools collected within one week of illness onset, making it unlikely that they arose from multiple mutations obtained during a prolonged infection. Since GII.4 noroviruses, the most homologous species to KL45 and T091, cause outbreaks associated with novel variants that displace others from previous years 
, it is possible that KL45 and T091 represent genetic variants that never became established.
A GIV.1 norovirus was detected in a stool obtained from a child in Entebbe, Uganda in 1976 (Hu/NoV/E22/Uganda/1976/GIV.1), making it the oldest known GIV.1 virus. In an evolutionary analysis, E22 clustered with other human GIV.1 noroviruses. GIV noroviruses are commonly associated with infections in animals, including dogs and lions, and are suggested to have arisen in humans from interspecies transmission due to the genetic similarity between human and animal strains 
. The 1976 date of collection for strain E22 indicates that GIV noroviruses have been circulating in humans on multiple continents for over 30 years.
Although this study contained a majority of GII noroviruses, we found a large number of GI.3 noroviruses as well. Rates of nucleotide evolution have not been reported previously for GI noroviruses, which circulate at lower frequencies 
. The nucleotide substitution rates for GI.3 and GI.1 noroviruses are similar for each clock model, which are consistent with traditional rates of evolution for RNA viruses 
, and with previously described rates for GII.3 and GII.4 noroviruses 
. The comparability in nucleotide evolutionary rates for GI and GII noroviruses indicates that nucleotide evolution is likely not responsible for the higher circulating frequencies of GII versus GI noroviruses.
Rotaviruses have maintained a dominance of the G1 genotype, and other minor genotypes have persisted in humans at lesser frequencies. Conservation of this limited pool of VP7 and VP4 genotypes suggests that multivalent vaccines may be necessary to obtain optimal protection against rotavirus gastroenteritis, and once formulated may persist in their effectiveness. For example, the oldest G9 rotaviruses belonging to Lineage I neutralize more contemporary strains of Lineages II and III, and therefore certain older rotavirus strains may be ideal candidates for attenuation and incorporation into vaccines 
. Further evidence for the vaccine potential of archival rotavirus strains includes the presence of contemporary G5 rotaviruses that are most closely homologous with older strains, including strain HK69 from this study. The overlapping representation of antigenic VP4 capsid genotypes in rotaviruses with distinct VP7 capsid genotypes, which was demonstrated in this archival study and is also currently observed, further supports rotavirus vaccine development strategies 
. It is important to note that both multivalent (RotaTeq: attenuated reassortant with human G1, G2, G3, G4, and P
antigenicity in a bovine backbone) and monovalent (Rotarix: attenuated human G1P
) vaccines have reduced the burden of rotavirus disease in countries where vaccination programs have been implemented 
. Although the composition of RotaTeq and Rotarix differ both in valency and source of viral strain (i.e. a reassortant with bovine backbone or an attenuated human strain), they have similar efficacies against severe rotavirus gastroenteritis, indicating that the relative importance of VP7 and VP4 antigenicity is unclear 
. Moreover, the first live, attenuated rotavirus vaccine, RRV-TV, composed of a closely homologous simian backbone strain, including a simian VP4, was efficacious against severe rotavirus gastroenteritis 
. Regardless of the specificity of vaccine formulations implemented to protect against the conserved VP7 and VP4 genotypes, additional surveillance data will need to be analyzed in the post-vaccine era to determine whether rotavirus evolution differs with the widespread introduction of vaccines 
On the other hand, noroviruses exhibit a large reservoir of genetic diversity, and our current understanding of this diversity may be limited, as evidenced by the presence of unique variants (and possibly new genotypes) detected in this cohort (KL45 and T091). Although KL45 and T091 have not been detected in current epidemiological surveys, strain C127 is an archival representative of the highly prevalent contemporary genotype GII.4, indicating that GII.4 noroviruses have maintained importance in the norovirus repertoire over more than 30 years. Thus, vaccination efforts against GII.4 noroviruses may be necessary. Several groups are currently investigating norovirus vaccine formulations for noroviruses and recent advances have been made particularly in GII.4-specific antibody epitope mapping 
. However, formulation of a cross-genotype, broad-spectrum norovirus vaccine is complicated by the lack of biological data, namely, it is unknown whether the designated VP1 capsid genotypes correlate with serotype specificity. Some insight was provided by a cross-challenge study in human volunteers, which demonstrated the importance of norovirus genogroups in protecting against disease, when administration of a GI.1 norovirus failed to confer protection upon cross-challenge with a GII.2 norovirus 
. This suggests that a norovirus vaccine may need to be multivalent, composed of at least three genogroups, which are most divergent among the noroviruses: GI, GII, and GIV.
The preservation of archival specimens by the LID over many years has allowed this retrospective characterization of norovirus and rotavirus molecular epidemiology and study of the evolution from specimens collected in various venues of the developing world in the late 1970’s. This investigation documents that the relative importance of noroviruses as agents of childhood gastroenteritis extends back more than 30 years and also reflects an early underestimation of norovirus incidence when the virus was discovered in the 1970’s. The characterization of rotavirus genotypes and viral lineages provides relevant archival information that supports current efforts towards implementing multivalent rotavirus vaccines.