In this study the genetic and phylogenetic analysis of the unusual P[14] RVA strains detected in 11 hospitalized children with rotavirus AGE from 2007 to 2021 in Greece was described. Multiple variants in the vaccine antigenic epitopes of the VP8* region was detected but with unknown significance. A vague evolutionary relationship between Greek human P[14] RVA strains and a Croatian fox P[14] RVA strain was indicated, which probably enhance the zoonotic origin of P[14] and the interspecies transmission from a wild animal to humans.
P[14] is a very unusual genotype and its frequency depends on the country. In our 14-year-study, its frequency was < 1%. P[14] is mostly combined with G8 RVA genotype worldwide. In Venezuela, before RVA vaccine implementation, the G8P[14] was the sixth most common RVA genotype, but after the vaccination period, the G8P[14] (2.4%) was the third most common RVA genotype detected after the G2P[4] and G1P[8] [20], [21]. In Japan, only one sample among 247 RVA positive samples was G8P[14]. This sample was detected in the winter of 2014 [22], in contrast with our P[14] samples that were almost all detected in the spring.
P[14] RVA strains of this study were combined with 3 different G genotypes: G8, G6 and G4. These genotypes, except for G4, have been previously detected sporadically (< 1%) in humans indicating zoonotic origin [23],[24],[25]. Their zoonotic origin are mainly from bovines and through interspecies transmission events they were transmitted to human [26], [27]. These genotypes are more common in countries that do not follow hygiene measures and come into contact very often with farm life as well as wildlife animals [28].
G8 and G6 are common in bovines [36], but G8 is also found in Oysters and shellfish [29], [30]. In Bangladesh, the predominant genotype (> 94%) among 200 diarrheic calves was the G6P[11] [29], while during 2017–2019 in India, the G6P[14] and G8P[14] were common RVA genotypes in bovines [30]. In Brazil, in 2010–2016, only the 3% of children < 3 years old with AGE were infected with G8P[4] [31], while in West Africa in 2010, an infant with diarrhea was infected with RVA G6P[14][32]. A study in Pakistan, in 2015, including 180 samples from children < 5 years old with AGE, G8 was among the most common RVA strains in infants [33], but in a study from Iran between the period 2017–2019, in 130 children < 15 years old with AGE, G8, G6 and P[14] were not detected [34]. Also, G8 strains have been reported by the EuroRotaNet as significant emerging strains in UK during 2008–2010 and 2018–2019 (EuroRotaNet, annual report 2019).
Recently, a new combination with P[14] was detected in Egypt in a 6-month-child with AGE, the G9P[14] [35]. The sequence of P[14] of this strain was highly similar to the old sequence EF554107 (95.45%) from Hungary [35]. This probably indicates that P[14] is conserved through time.
Most of the RVA phylogenetic and genetic analysis in feces of humans and animals with gastroenteritis, had shown that P[14] has zoonotic origin and specifically from bovines, rabbits, etc. A study from Japan has shown that the P[14] genotype resembles a lot with cow strains [22]. This is the first report, in which a P[14] RVA phylogenetic relationship between fox and human was revealed. This finding highlights the interspecies transmission through wild animals and humans.
So far, there is only one study that tested for RVA infection the foxes’ feces in Croatia, where the foxes live near to urban areas. They discover that one red fox (Vulpes vulpes) was infected with a G8P[14] RVA strain in 2018 [36]. Therefore, animals of wildlife like foxes may be potential reservoirs for unusual strains.
The use of next generation sequencing (NGS) can help to elucidate possible reassortment events, the number of these events and the potential zoonotic origin of a strain. The susceptibility of P[14] in reassortment events has been studied around the world. For example, the genomic analysis using NGS of two G8P[14] strains isolated from two children with AGE in Italy in 2012 revealed reassortment events through human and sheep RVA strains [37]. In a Slovenian study, a RVA strain, isolated from a 1-year-child with severe AGE in 2009, genotyped using NGS revealing a zoonotic origin of the strain (G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3) [38]. In another study in Hungary, the genome analysis of a G8P[14] from a 4-year-child with AGE in 2001 using NGS revealed that more than one reassortment event occurred in this strain (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) [39]. In Honduras, the use of NGS in a RVA G10P[14] strain isolated from a 30-month-child with severe AGE also reveals multiple reassortments events enhancing the zoonotic origin of this strain and its many potential reservoirs [40]. All these strains were pathogenic to humans but the exact molecular factor leading to severe AGE remains unclear. Larger studies or meta-analyses should be conducted to clarify these factors.
It is known the significant role of the viral proteins VP8* (derived from VP4) and VP7 in host cell attachment binding to cellular receptors containing sialic acid (SA) or histo-blood groupantigens (HBGAs) or to cell surface components like sialoglycans [11],[41],[42]. In the stage of endocytes of the RVA, the VP4 undergoes structural changes displaying its hydrophobic sites [43]. Specifically, the protein VP4 contains three highly conserved trypsin cleavage sites at Arginine 231, 241 and 247 that divide it into two polypeptides, VP5* and VP8*, the length of which depends on the digested site [44]. This cleavage process of VP4 provokes high infectivity [45]. In all our samples, these regions are conserved. However, our samples carried many variants near to significant sites or within to antigenic epitopes. In contrast to our results, analysis of the antigenic epitopes in two G8P[14] strains in Italy shows that the sequences were conserved [37]. Further studies regarding the mutagenesis of these strains and the role of this variants in pathophysiology and severity of the disease should be conducted.
A limitation of our study is the weakness to investigate the complete genotype constellation of these P[14] strains using NGS methodologies in order to reveal possible reassortment events. This is the first molecular report of the unusual P[14] RVA strains detected in Greece, which focuses on phylogenetic analysis and enhances their potential zoonotic origin, while previous studies have described the genotype distribution of common and less common genotypes circulating in Greek children [19],[46],[47]. The continuous surveillance of RVA genotypes distribution and genomic evolution is crucial to understand better this virus, the disease that causes and the potential need to develop new RVA vaccines.