Genomic Constellation of Human Rotavirus G8 Strains in Brazil over a 13-Year Period: Detection of the Novel Bovine-like G8P[8] Strains with the DS-1-like Backbone

Rotavirus (RVA) G8 is frequently detected in animals, but only occasionally in humans. G8 strains, however, are frequently documented in nations in Africa. Recently, an increase in G8 detection was observed outside Africa. The aims of the study were to monitor G8 infections in the Brazilian human population between 2007 and 2020, undertake the full-genotype characterization of the four G8P[4], six G8P[6] and two G8P[8] RVA strains and conduct phylogenetic analysis in order to understand their genetic diversity and evolution. A total of 12,978 specimens were screened for RVA using ELISA, PAGE, RT-PCR and Sanger sequencing. G8 genotype represented 0.6% (15/2434) of the entirely RVA-positive samples. G8P[4] comprised 33.3% (5/15), G8P[6] 46.7% (7/15) and G8P[8] 20% (3/15). All G8 strains showed a short RNA pattern. All twelve selected G8 strains displayed a DS-1-like genetic backbone. The whole-genotype analysis on a DS-1-like backbone identified four different genotype-linage constellations. According to VP7 analysis, the Brazilian G8P[8] strains with the DS-1-like backbone strains were derived from cattle and clustered with newly DS-1-like G1/G3/G9/G8P[8] strains and G2P[4] strains. Brazilian IAL-R193/2017/G8P[8] belonged to a VP1/R2.XI lineage and were grouped with bovine-like G8P[8] strains with the DS-1-like backbone strains detected in Asia. Otherwise, the Brazilian IAL-R558/2017/G8P[8] possess a “Distinct” VP1/R2 lineage never previously described and grouped apart from any of the DS-1-like reference strains. Collectively, our findings suggest that the Brazilian bovine-like G8P[8] strains with the DS-1-like backbone strains are continuously evolving and likely reassorting with local RVA strains rather than directly relating to imports from Asia. The Brazilian G8P[6]-DS-1-like strains have been reassorted with nearby co-circulating American strains of the same DS-1 genotype constellation. However, phylogenetic analyses revealed that these strains have some genetic origin from Africa. Finally, rather than being African-born, Brazilian G8P[4]-DS-1-like strains were likely imported from Europe. None of the Brazilian G8 strains examined here exhibited signs of recent zoonotic reassortment. G8 strains continued to be found in Brazil according to their intermittent and localized pattern, thus, does not suggest that a potential emergence is taking place in the country. Our research demonstrates the diversity of G8 RVA strains in Brazil and adds to the understanding of G8P[4]/P[6]/P[8] RVA genetic diversity and evolution on a global scale.

The sequences obtained were aligned and converted to proteins using reference sequences to identify the open reading frame and subsequent protein alignment. With the sequences created in this manner, we separated each case into its own FASTA file and proceeded with the modeling in MODELER 10.4 and SwissModel, evaluating them with DOPE scores [44,45]. The sequences were then evaluated according to the PDBSum GEN-ERATE scores [46] and structurally aligned by the PyMod modeler module (SAlign) from the Pymol 2.5 (https://pymol.org/2/). The models were then treated on the MolPorbity website (http://molprobity.biochem.duke.edu/) to check clashes and bumps. The final configurations were then evaluated on the Immune Epitope Database (IEDB) website (https://www.iedb.org/) in the DISCOTOPE and ELlipro modules to investigate discontinuous epitopes and predict antibody binding, both using 3D structures for the losses.
To investigate the genetic relatedness and potential origin of the Brazilian G8P [4]/P[6]/ P [8] strains, the 11 gene segments were analyzed phylogenetically. The phylogenetic relationship was inferred by the maximum-likelihood method, using reference RVA strains from humans, vaccines, cows, goats, foxes, alpacas, bats, pigs, camels, sheep, roe deer, horses, cats, vicuñas, simians, dogs, rabbits, antelopes, guanacos, rats, llamas and lambs available at the GenBank database. Sequences from Brazil and South America were also included in the analysis.
The sequences of the two Brazilian bovine-like G8P [8] strains with the DS-1-like backbone strains were analyzed to elucidate the origin of these strains, whether they are derived from one specific bovine-like G8P [8] DS-1-like reference strain, or whether they are reassortment strains. The VP7, VP4 and NSP2 genes of strains IAL-R193 and IAL-558 clustered together exclusively with bovine-like G8P [8] strains with the DS-1-like backbone circulating in Asian countries (nucleotide sequence identities of 99-100%, 98-99% and 99.5-100%, respectively) ( Figure 1A,B,J). Considering the VP7 gene in particular, the two bovine-like G8P [8] strains with the DS-1-like backbone clustered together with the majority of human RVA G8P [8] DS-1-like reference strains and with the bovine RVA/Cowwt/IND/68/2007/G8P [14] ancestor, reinforcing the hypothesis that these newly discovered bovine-like G8P [8] strains with the DS-1-like backbone have an animal origin ( Figure 1A). Considering the VP4 gene, the Brazilian bovine-like G8P [8] strains with the DS-1-like backbone detected here grouped into Lineage III, as expected. After 2003, virtually all globally circulating P [8] strains belonged to Lineage III ( Figure 1B).
The comparative VP7 sequence analysis revealed that the bovine RVA G8P [1] NGRBg8/ 1998 strain detected in Nigeria and the human RVA G8P [6] DS-1like strains reported in the present investigation share nucleotide identities varying from 95.6% to 95.9%, highlighting G8 strains' potential ruminant ancestry ( Figure 1A). In addition, genetic analysis of the VP4 gene revealed that the African straw-colored fruit bat (Eidolon helvum) RVA G25P [6] 4852/2007 strain detected in Kenya [22] also clustered inside Lineage I-a together with the six Brazilian human G8P [6] DS-1-like strains reported here, displaying 94.1-94.9% of nucleotide similarity among them ( Figure 1D).

Modeling of the VP7 Gene
In order to better understand the differences in amino acid composition and the antigenic characteristics of human and animal G8 strains, the amino acid sequences of the VP7 antigenic region were examined. The results were ranked according to the DOPE scores and graphed accordingly (Supplement S6).
Regarding the reference structures of human origin, the distance matrix shows similarities above 94% for the cases identified in this study. From the point of view of amino acids, similar values (91.46 up to 100%) were detected in the bovine samples as well. These similarities in themselves suggest that there is little difference in the antibody recognition of this protein in the human immune system. However, to verify this correctly, we made predictions regarding the interactions with antibodies and with discontinuous epitopes, which could emerge from the differences, however small they were. Structurally, the proteins diverged very little from the human and bovine references, as shown in Table 2, and structural alignment (Figure 2). Furthermore, the results for the detection of discontinuous epitopes and antibody interaction suggest overlap in all cases, thus excluding the possibility of differential epitopes based on observed differences. Table 2.

IAL
the DS-1-like backbone, detected in distinct Brazilian regions. Whole-genotype characterization is crucial in tracking the emergence of novel RVA strains and understanding their evolution [59].
Strains exhibiting the G8 genotype are considered rare or uncommon [5,20]. The low frequency of RVA G8 infections detected in the present study (0.6%; 2007-2020) agreed with data previously described in Brazil in both pre-(1%; 2005) [27] and post-RVA vaccine eras (0.5%; 2007-2012) [47]. They were also similar to that observed in other studies carried out in Croatia (0.1%; [60] and Thailand (0.6%;2003 [61]. A relatively high prevalence of G8 RVA strains has, for decades, been commonly observed in African countries [62][63][64][65][66]. Nevertheless, the increase in G8 detection outside Africa, such as in Asia, the Middle East and European countries [15,67,68] may imply that G8 strains are emerging across the globe and that this specific genotype should be carefully monitored. Oscillatory trends in the incidences of RVA genotypes are widely observed phenomena [3,5,29], and the emergence of the G8 could be explained by vaccine-induced genotypes and irregular RVA immunization schedules, or both [69,70]. Continued RVA surveillance is vital to better understand the contemporaneous role of G8 strains within human populations. In the present investigation, G8 strains followed their sporadic and confined pattern of detection in Brazil, thus not suggesting that a potential emergence is taking place in the country. As is usual among RVA strains, the putative VP7 N-linked glycosylation site was located at amino acid (aa) 69 in Brazilian G8P [4]/P [6]/P [8] DS-1-like strains [71]. Additionally, like the majority of bovine and human G8 strains and Brazilian G8 strains reported here possessed a second glycosylation site at aa 238 [33,72]. Glycosylation of residue 238 has been observed to decrease the neutralization of animal G11 RVA strains by hyperimmune sera and MAbs, which may have broad implications for immunogenicity [73]. Inside the main antigenic site, region D (aa 143-152), an amino acid change at position 145 N→S in G8P [4] DS-1-like strains and at position 146 A→T in G8P [6] DS-1-like strains took place. Region E (aa 207-220), which is spatially close to region D, contains the amino acid substitution at position 218 I→V in G8P [4] DS-1-like strains and at position 218 V→I in G8P [6] DS-1-like strains [48]. The Brazilian G8P [8] DS-1-like strains did not exhibit amino acid substitutions in the D and E regions. Antigenic analyses using the VP7 gene did not reveal any distinctions between the epitopes of the G8 strains either from human or animal origin.
The VP4 spike protein is cleaved by trypsin to produce the polypeptides VP8* and VP5*, which are needed to activate infectivity [74,75]. The Brazilian G8P [4]/P [6]/P [8] DS-1like strains preserved the potential VP4 arginine cleavage sites (230, 240, and 246), assuring infectivity. The four proline residues (68, 71, 224 and 225) are also conserved. Given that proline is known to cause three-dimensional structural distortion, these conserved prolines may have a significant impact on the conformation of the VP4 [75]. A limitation of the current study was the failure to obtain entire sequences of the VP4 gene, impairing protein modeling analyses from being conducted.
Atypical reassorted bovine-like G8P [8] strains with the DS-1-like backbone emerged during the 2013/2014 seasons in Southeast Asia [17,49,50,76], spreading to Europe and South America. They were also recently reported in the Czech Republic (2016-2018) [15] and Argentina (2018) [51,77]. Of the two Brazilian bovine-like G8P [8] strains with the DS-1-like backbone reported here, one (IAL-R193/2017) was acquired by a 7-month-old male patient in the city of Goiânia, Midwestern region. The other sample (IAL-R558/2017) was collected from a 4-month-old male child in the city of São Paulo, Southeastern region. These data indicate that the bovine-like G8P [8] DS-1-like strains have circulated in different Brazilian regions at the same time. Moreover, based on timeline detection data, it could be speculated that atypical bovine-like G8P [8] strains with the DS-1-like backbone reached the South America through Brazil (2017), then disseminated to other nations such as Argentina (2018) [51,77]. The route of bovine-like G8P [8] strains with the DS-1-like backbone spreading across the globe is tricky to be recognized, but globalization is probably the key point of RVA strain traffic from one continent to another [15].
An important issue to be highlighted is the fact that bovine-like G8P [8] strains with the DS-1-like backbone did not remain in circulation in the Brazilian population, since they were not detected in the subsequent years of the surveillance (2018 to 2020). This is different to what was observed with the equine-like G3P [8] DS-1-like strains [13]. Therefore, it can be suggested that bovine-like G8P [8] strains with the DS-1-like backbone do not achieve the fitness required to become a successful human pathogen in Brazil, as observed in Asian countries [17,50].
On the one hand, the VP1 R2 genotype identified in the bovine-like G8P [8] strain with the DS-1-like backbone detected in Midwestern Brazil (IAL-R193/2017) belong to lineage XI and grouped together with the most recent bovine-like G8P [8] strains with the DS-1-like backbone detected since 2013 in Asia, including Japan, Vietnam, Thailand and Korea [17,49,50]. On the other hand, the VP1 R2 genotype recognized in the bovine-like G8P [8] strain with the DS-1-like backbone detected in Southeastern Brazil (IAL-R558/2017) possessed a distinct R2 lineage never previously described and grouped apart from any of the DS-1-like reference strains. Therefore, the bovine-like G8P [8] strains with the DS-1-like backbone detected in Midwestern and Southeastern Brazil in 2017 may have been introduced into the country from distinct pools of co-circulating bovine-like G8P [8] strains with the DS-1-like backbone. Intra-genotypic variability and distinct genotypic lineage constellations of the bovine-like G8P [8] strains with the DS-1-like backbone have been previously reported [78][79][80].
Additionally, the phylogenetic analysis of the NSP genes has demonstrated that the Brazilian bovine-like G8P [8] strains with the DS-1-like backbone clustered together with novel DS-1-like G1/G3/G9/G8P [8] strains detected in Asia, Europe and Americas [11][12][13][14][15][16][17], as well as with classical G2P [4] strains circulating in Australia in 1999 and in Japan in 2001 [43,81]. These findings collectively imply that the origin of the Brazilian bovine-like G8P [8] strains with the DS-1-like backbone is probably not directly related to importation from Asia, but rather that the atypical bovine-like G8P [8] strains with DS-1-like backbone continue to evolve, most likely through reassortment with regionally prevalent RVA strains. Over time, a globally co-circulating pool of different bovine-like G8P [8] strains with the DS-1-like backbone should be expected due to its natural evolution and/or rearrangements with local RVA strains.
G8P [4] DS-1-like strains are found mainly in Africa (especially Malawi) and sporadically reported in Europe, Asia and the Americas, including in Brazil [21,25,52,62,63,[82][83][84]. The four Brazilian G8P [4] DS-1-like strains revealed by phylogenetic research had nearly identical sequences when all 11 gene segments were taken into account. Additionally, a close relation was observed between Brazilian G8P [4] DS-1-like strains and two European G8P [4] strains for all genes investigated: the GER1H-09 isolated in Germany in 2009 [52] and the SS65 reported in Italy in 2011 [55]. Together, these findings suggested that the Brazilian G8P [4] DS-1-like strains were probably imported from Europe rather than being African-born. The VP7 gene of the Brazilian G8P [4] DS-1-like strains was the only genomic segment that, besides these two European G8P [4] strains, also clustered together with some Brazilian G8P [4] strains detected between 2010 and 2011 [21]. The origin of Brazilian G8P [4] DS-1-like strains described here may have involved reassortment events with locally G8 RVA circulating strains.
All four Brazilian G8P [4] DS-1-like strains were identified in 2010 in the city of Brasilia (Brazil's Capital), located within Goiás state (GO). Silva-Sales et al. [21] recently reported the full genome characterization of another four G8P [4] DS-1-like strains, also detected in 2010, but in the state of Tocantins (TO). Goiás and Tocantins are bordering states situated in the central region of the country, which is home to the Brazilian savanna biome. A significant finding could be drawn from this context. NSP1, NSP2 and NSP3 gene phylogenetic trees have shown that Brazilian Goiás G8P [4] DS-1-like strains and the Brazilian Tocantins G8P [4] DS-1-like strains, published previously [21], clustered in different lineages, suggesting genetic variety among Brazilian G8P [4] DS-1-like strains, which were essentially discovered at the same time and location. Collectively, the genomic analysis revealed that the Brazilian G8P [4] DS-1-like strains appeared to have undergone genetic reassortment events with both locally and globally circulating strains.
A potential interspecies transmission based on multiple reassortment events between artiodactyls, ruminant and human RVA strains were suggested for G8P [4] DS-1-like RVA strains detected in Asia [82,83]. The genetic analysis of the Brazilian DS-1-like G8P [4] RVA strains conducted here did not indicate a recent zoonotic origin, following previous phylogenetic investigations performed in European countries and Brazil [21,52,55]. Nevertheless, it is worth mentioning that a certain link between human and animal DS-1-like G8P [4] RVA strains does probably exist, as we recognized genetic relatedness of human DS-1-like G8P [4] VP7, NSP2 and NSP4 gene segments to those of bovine, sheep and dog RVA strains, respectively, attempting to hypothesize footprints of interspecies transmission events. More in-depth molecular analysis of DS-1-like G8P [4] RVA strains is hampered by a lack of genome sequencing data of RVA strains circulating in animals, and this is especially the case for Brazil.
Significant epidemiological relevance has been placed on the G8P[6] DS-1-like genotype in Africa [57,62,63,85]. The Brazilian G8P [6] DS-1-like RVA strains reported here were detected from two different populations: four strains were obtained during an outbreak affecting Brazilian native children in the city of Dourados (Midwestern region) in 2009 [26] and two strains were acquired from children with acute gastroenteritis during the epidemiological survey in São Paulo city (Southeastern region) in 2010. The six G8P [6] DS-1-like strains were genetically similar to each other and clustered together in all 11 gene segments, therefore suggesting that, during those two years, the same G8P [6] DS-1-like strain was circulating throughout different parts of Brazil.
The Brazilian G8P [6] DS-1-like showed no evidence of recent zoonotic reassortment events, but genetic similarity between the African bat G25P [6] RVA strain and human G8P [6] RVA strains have been reported [22,26]. Genetic studies point to a possible porcine origin for the P [6] genotype [86]. The paucity of fresh conclusions drawn from the phylogenetic studies performed here is due to the dearth of information on animal P [6] strains. It is important to mention that four G8P [6] DS-1-like strains characterized here were characterized by Brazilian native children. It is well known that indigenous communities live in proximity to animals, sustaining the continuous exposure to potential interspecies transmission of RVA strains [26]. These data underscore the need for increased animal RVA molecular surveillance and attention to the value of a One Health strategy in the field of RVA research.
In conclusion, this is a pioneer study analyzing the complete constellation of G8P [4], G8P [6] and G8P [8] RVA strains detected in Brazil, as well as the first report of the novel bovine-like G8P [8] strains with the DS-1-like backbone circulating in the country. The genetic information obtained here has the potential to provide the basis for monitoring variations in the molecular composition of G8 RVA strains circulating in the Brazilian human population. Our findings highlight the variety of G8 RVA strains in Brazil and also contribute to the knowledge of G8P [4]/P [6]/P [8] RVA genetic diversity and evolution from a global perspective.