Introduction

Acute gastroenteritis (AGE) is characterized by the presence of diarrhea and/or vomiting, possibly accompanied by fever, abdominal pain, and dehydration [1]. It is responsible for approximately 10% of pediatric mortality worldwide [2]. Depending on the country, rotaviruses (RVs) are among the most common pathogens causing AGE [3, 4]. Although several groups of rotaviruses (A-D and F-J) have been identified, only A-C and H can infect both humans and animals. Among the human rotaviruses, rotavirus group A (RVA) is the most common [5, 6].

Neonates and children under the age of 5 years old are the most frequently infected with RVA, and according to reports of the Global Burden of Disease, RV infection causes > 250 million episodes of diarrhea and > 120 thousand deaths annually [7]. However, more than ten years after the implementation of the RVA vaccines Rotarix® (GlaxoSmithKline, Rixensart, Belgium, RV1) and RotaTeq® (Merck & Co, Lyon, France, RV5), a 40.6% decrease in the mortality rate has been observed in children < 5 years old [8,9,10].

RVA belongs to the family Reoviridae, and its genome consists of 11 double-stranded RNA segments, which encode six structural (VP1-VP4, VP6, and VP7) and six non-structural viral proteins (NSP1-NSP6) [11]. For the genotyping of RVA, an international classification system has been established based on the sequences of two viral proteins: VP4 (protease-sensitive protein) for the P genotype and VP7 (glycoprotein) for the G genotype [12]. These two proteins form the outer capsid of the virus, participate in host cell attachment and penetration, and also contain neutralization epitopes [13].

To date, at least 42 G and 58 P RVA genotypes have been identified in humans and animals (https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg). Several domestic animals such as dogs, cats, cows, and pigs as well as non-domestic animals such as bats, rodents, and birds can be infected with RVs [14]. Although more than 80 G and P combinations of RVA have been detected, the most common RVA genotypes circulating in humans worldwide are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] [15]. Unusual genotypes such as P[14] and mixed types (with more than one G and/or P type) have also been reported. Strains carrying the unusual P[14] genotype are sporadically detected in humans and usually show a zoonotic origin [16]. It is not unprecedented that human-to-animal contact may contribute to interspecies transmission, as appears to be the case for P[14] strains [17].

The aims of this study were to describe the epidemiology of unusual RVA genotypes isolated from children presenting with AGE from 2007–2021 and to examine the molecular characteristics of human P[14] RVA strains.

Materials and methods

Study design

This was a multicenter study conducted at the Infectious Diseases Laboratory of the Choremeion Research Laboratory, “Aghia Sophia” Children’s Hospital, and involved the analysis of 4427 fecal samples from children ≤ 16 years old with AGE from 20 Greek paediatric hospitals during the period 01/2007–12/2021.

Children who were admitted to “Aghia Sophia” Children’s Hospital, a 750-bed tertiary paediatric hospital in the metropolitan Athens area, or to 19 other paediatric hospitals, covering the majority of the national paediatric population, and fulfilled the criteria for AGE were tested for rotavirus infection using a rapid immunochromatographic test (VIKIA® Rota-Adeno test, bioMérieux, Lyon, France). Samples that were positive for RVA were sent within a 10-day period to the Infectious Diseases Laboratory of the Choremeion Research Laboratory in Athens for RVA genotyping according to EuroRotaNet’s guidelines (https://www.eurorotanet.com/project-information/documents-and-methods/). The study protocol was approved by the scientific and bioethics committee of “Aghia Sophia” Children’s Hospital (No. 6261).

Data collection

Demographic and clinical data were also collected from children infected with unusual RVA, including age, sex, residence, symptoms of AGE (diarrhea, vomiting, fever, and dehydration), laboratory data from blood samples, days of hospitalization, and RVA vaccination status. Laboratory data included values for potassium (K+), sodium (Na+), calcium (Ca2+), chlorine (Cl-), C-reactive protein (CRP), urea, creatinine, white blood cells (WBC), polymorphonuclear leukocytes, and lymphocytes that were requested in the context of hospitalization for AGE.

Reverse transcription and gene amplification

Fecal samples were prepared as a 10% suspension with Stool Transport and Recovery (S.T.A.R.) buffer (Roche Diagnostics, Basel, Switzerland) and 7% chloroform for the extraction of viral genomic RNA, employing a MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics, Basel, Switzerland) on a MagNA Pure Compact instrument according to the manufacturer’s instructions. Viral RNA was stored at -80°C or used immediately.

Synthesis of cDNA was carried out using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) and multiplex semi-nested PCR for the amplification of the VP7 and VP4 regions were conducted using GoTaq DNA Polymerase (Promega, Madison, Wisconsin, USA) and specific primers according to European Rotavirus Detection and Typing Methods [18]. The PCR products were initially characterized as G (G1-4, G8-10, and G12) and P (P[4], P[6], P[8], and P[9]-[11]) types according to the PCR product size, using 2% agarose gel electrophoresis with a 50-bp DNA ladder (N3236S; New England Biolabs, Massachusetts, USA) and ethidium bromide staining.

Sequencing

Further genotyping of RVA strains that were not genotyped by agarose gel electrophoresis was carried out by Sanger sequencing of the VP7 and VP4 genes using a BigDye Terminator v3.1 Cycle Sequencing Kit on an Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The electrochromatographic data from sequencing were further analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Phylogenetic analysis

Phylogenetic evolutionary analysis of P[14] RVA strains was performed using MEGA 11 software (Molecular Evolutionary Genetics Analysis; www.megasoftware.net). All P[14] sequences from human and animal strains were obtained from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and Virus Variation (https://www.ncbi.nlm.nih.gov/genome/viruses/variation/) databases. Multiple sequence alignments were performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation) software. A model/method test was performed. Phylogenetic trees were constructed using the maximum-likelihood method, the Tamura 3-parameter model, and bootstrap resampling with 1000 replicates. The substitution model used was the maximum composite likelihood model. Nucleotide sequence similarity was analyzed using BLAST. The sequences determined in this study were deposited in the GenBank database with accession numbers ON009343-44, ON004940, OM912818-19, OM303087, OM891773, OM829817, OM303090, OP183207-08, and OQ606801.

Results

Genotyping and study population

In this study, 4427 fecal samples from children ≤ 16 years with AGE who were infected with an RVA strain were included. Genotyping showed that 62 (1.4%) children with a median age of 18.7 (IQR: 37.3) months had been infected by an unusual G (G6: 16.1%; G: 11.3%; G10: 4.8%) or P (P[6]: 17.7%; P[9]: 45.2%; P[10]: 9.7%; P[11]: 1.6%; P[14]: 19.4%) genotype. The majority of these children belonged to the age group of 13–48 months (25/62, 40.3%), 67.7% (42/62) were male, 25.8% resided in rural areas, and 63% (39/62) were of Greek origin. None of them had been vaccinated for RVA. Their AGE symptoms were diarrhea (77.4%; 48/62), vomiting (58.1%; 36/62), fever (66.1%; 41/62), and moderate dehydration (40.3%; 25/62). Their laboratory values were within the normal range, except for CRP, which was slightly elevated (mean ± SD: 17.2 ± 28.7). Their median (IQR) days of hospitalization was 3.0 (2.0) days.

In total, P[14] (19.4%; 12/62) was the second most common unusual genotype after P[9] (28/62; 45.2%). Three different genotype combinations with P[14] were detected, G8P[14] (41.7%; 5/12), G6P[14] (41.7%; 5/12), and G4P[14] (16.6%; 2/12). The genotype combinations of unusual RVA strains detected in this study are shown in Table 1. The median (IQR) age of children with P[14] RVA infection was 50.6 (52.12) months, 58.3% (7/12) were males, 83.4% (10/12) were of Greek origin, 8.3% (1/12) were of Afghan origin, 8.3% (1/12) were of Albanian origin, and 33.3% (4/12) resided in rural areas. Their symptoms included diarrhea (10/12; 83.3%), vomiting (8/12; 66.6%), fever (8/12; 66.6%), and moderate dehydration (7/12; 58.3%). Specifically, diarrhea or vomiting was present in 58.3% (7/12), and both symptoms were present in 50% (6/12) of the patients. The median (IQR) days of hospitalization was 3.33 (2.07).

Table 1 Detected G and P combinations in unusual rotavirus group A strains
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Regarding the laboratory values for children infected with P[14] RVA infection, it was found that all of the children had slightly elevated CRP (mean ± SD: 18.2 ± 19.1 mg/l; normal values, 0–1 mg/l for children ≤ 1 year old and 1–10 mg/l for those > 1 year old), and one child showed electrolytic disturbances with low potassium (K+: 3.4 mmol/l; normal values: 3.5–5.5 mmol/l) and sodium (Na+: 133 mmol/l; normal values, 135–150 mmol/l). All of the other values were normal.

Annual and seasonal distribution

An increase in the annual distribution of unusual RVA strains was observed in 2019–2021, in the COVID-19 pandemic period, with the highest circulating levels (14.1%; 12/85) in 2020 (Fig. 1). Genotypes with a prevalence > 1% were detected: P[6] (1.3%) in 2011, P[9] (1.7%) and P[10] (2%) in 2019, P[9] (9.4%), P[14] (2.4%), G8 (2,4%), and G10 (1.2%) in 2020, and P[9] (1.8%), P[14] (1.8%), and G6 (2.3%) in 2021. The seasonal peak was during spring (March to May: 37.1%; 23/62) and autumn (September to November: 33.9%; 21/62), whereas the prevalence was lower in winter (20.9%; 13/62) and summer (8.1%; 85/62).

Fig. 1
figure 1

Annual distribution of total (n = 4427) and unusual (n = 62) rotavirus group A (RVA) cases from 2007–2021

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Regarding P[14] strains, the annual distribution was as follows: 2.7% (1/37) in 2007, 0.9% (4/439) in 2010, 0.4% (2/500) in 2012, 2.4% (2/85) in 2020, and 1.8% (3/171) in 2021. In the last two consecutive years, during the COVID-19 pandemic period, an emergence of this strain was observed (1.6%; 4/256 in all RVA samples and 41.7%; 5/12 in P[14]) after 8 years of absence. Their seasonal peak occurred during the spring months, March to May (91.7%; 11/12). P[14] genotypes were detected in 12 children from six different children’s hospitals. The geographical distribution of this genotype in Greece was as follows: eight genotypes were isolated in Athens (three hospitals), two in Karditsa, one in Larisa (a previously reported genotype [19]), and one in Kalamata.

Molecular characterization of P[14] strains

From the 12 samples from children with P[14] RVA infection, a portion of the VP4 gene was sequenced. This part includes almost all of the VP8* region (amino acids 1-231), the fragment between the VP8* and VP5* regions, and the start of the VP5* region (up to amino acid 264). The P[14] sequences of these strains were aligned and compared with all recorded P[14] RVA (human and animal) sequences (n = 94).

Many synonymous and missense gene variants were detected. Three of the missense variants were novel: V61I, Q62S, and Q127R. The variants V61I and Q62S, detected in RVA/Human-wt/GR/20210521/3005/2021/G8P[14] (OM303090.1) and RVA/Human-wt/GR/20210605/2974/2021/G6P[14] (OM829817.1), respectively, are in the VP8* region, which interacts with the viral protein VP6 [11] [20]. The mutation V61I involves the replacement of a hydrophobic amino acid with another one, and the mutation Q62S represents a replacement of a hydrophilic amino acid with a hydrophobic neutral amino acid. The novel VP4 variant Q127R was detected in the strain RVA/Human-wt/GR/20100409/517/2010/G6P[14] (OM912819.1). This variant is located in the VP8* spike head, in the lectin-like, globular domain, and involves the replacement of one hydrophilic amino acid with another hydrophilic amino acid.

When comparing the sequences of the P[14] RVA strains from this study with those of the antigenic epitopes of the VP8* region of the Rotarix and RotaTeq vaccine strains [21], many different variants were detected (Table 2). The amino acid D100 in the 8 − 1 epitope appears to be conserved, except in the strain RVA/Human-wt/GR/20120422/19–38/2012/G6P[14], which carries the homozygous D100N variant, which involves the replacement of a negatively charged amino acid, aspartic acid (D), with an uncharged amino acid, asparagine (N), which has a similar size. This position is probably important because it is next to amino acid 101, which binds to sialic acid on the host cell. A search of the other 94 recorded P[14] sequences showed that only three carried this variant: RVA/Human-wt/JPN/Tokyo/12-1375/2012/G8P[14], RVA/Human-wt/BEL/B4106/2000/G3P[14], and RVA/Human-wt/BEL/BE5028/2012/G3P[14]. The frequency of this variant among P[14] strains (n = 106) was thus estimated to be 3.8%.

Table 2 Variants e in antigenic epitopes of the VP8* region of P[14] rotavirus group A (RVA) strains (n = 12)
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The strain RVA/Human-wt/GR/20210516/3096/2021/G6P[14] also carried three variants when compared to the vaccine strains and the other strains from this study: N/I89T, N/S113P, and R/N183D (Table 2). Although these amino acid positions are probably less conserved, none of them are novel. The variant N/I89T is in the 8 − 4 epitope, and it was previously reported in three animal strains (rabbit and antelope) and five human strains in Europe and North America from 1989. The variant N/S113P is located within the 8 − 3 epitope and has previously been reported only in two animal strains from America (RVA/Guanaco-wt/ARG/Chubut/1999/G8P14) and Africa (RVA/Goat-wt/MAR/Ch_S44/2014/P14). The variant R/N183D, located in the 8 − 2 epitope, was found in three human strains from Europe (RVA/Human-wt/ITA/PA77/2002/P14, and RVA/Human-wt/ESP/Sp813/2007/P14, RVA/Human-wt/BEL/B10925/1997/G6P14). The frequency of these variants among the P[14] strains (n = 106) was 8.5%, 2.8%, and 3.8%, respectively.

Position 113 seems to be variable, since one of our 12 P[14] strains RVA/Human-wt/GR/20200422/2887/2020/G8P14, carried the variant N/S113Q, which is also present in 27 animal and human P[14] strains (frequency: 26.4%). Despite the physicochemical differences between all these amino acids, their contribution to transmissibility and morbidity remains unknown.

Phylogenetic analysis of P[14] strains

Phylogenetic analysis was performed using 106 VP4 gene sequences (94 from public databases and 12 from this study) to clarify their genetic relationships and evolutionary history (Fig. 2). Nine of the strains from this study may have a European ancestor, three of which probably originated from an RVA strain that was detected in a wild animal. One may have originated in Egypt, another in Vietnam, and another in either Hungary or India.

Fig. 2
figure 2

Phylogenetic tree of P[14] rotavirus group A (RVA) strains based on partial VP4 gene sequences. Samples from this study are indicated by a green circle. Sequences obtained from the GenBank database are labeled with their accession number, two or three letters of the country of origin, the year of isolation, and their genotype. The maximum-likelihood method, the Tamura 3-parameter model, and a bootstrap test of 1000 replicates were used to infer the phylogeny. Bootstrap values above 70% are shown next to branches. The bar represents 0.050 substitutions per nucleotide position. HRV, Croatia; HUN: Hungary; ITA: Italy; FRA: France; KEN: Kenya; EGY: Egypt; IND: India; ESP: Spain; BEL: Belgium; MAR: Morocco; GHA: Ghana; ARG: Argentina; AUS: Australia; USA: United States of America; JPN: Japan; TUN:Tunisia; BRB: Barbados; THA: Thailand; DEU and GER: Germany; TWN: Taiwan; VNM: Vietnam; PER: Peru; GTM: Guatemala; HON: Honduras; GR: Greece; ZAF: South Africa; CHN: China, SLO and SVN: Slovenia

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Phylogenetics showed that the VP4 amino acid sequence of strain RVA/Human-wt/GR/20070101/057/2007/G8P[14] is 97% related to that of the 2004 Italian strains RVA/Human-wt/ITA/PR1300/2004/G8P[14] and RVA/Human-wt/ITA/PR/1300/04/2004/P[14], with 99% nucleotide sequence identity due to the presence of seven synonymous and two missense mutations as shown using BLAST. The strain RVA/Human-wt/GR/20100331/50016/2010/G4P[14] was 77% related to the Spanish strain RVA/Human-wt/ESP/Sp1524/2009/P[14], with 99% nucleotide sequence identity (five synonymous and one missense mutation). Both strains also were 77% related to a farm animal strain from Spain, RVA/Sheep-tc/ESP/OVR762/2002/G8P[14]. The VP4 of strain RVA/Human-wt/GR/20100406/5–12/2010/G4P[14] is less than < 70% related to other strains.

The VP4 of Greek strain RVA/Human-wt/GR/20100407/60036/2010/G6P[14] is 99% evolutionary related to that of Italian strain RVA/Sewage-wt/ITA/BA-M11-07/2011/P[14], which was isolated from sewage in 2011, with only three variants (also 99% nucleotide identity), all of which are synonymous substitutions. The ancestor of RVA/Human-wt/GR/20100409/517/2010/G6P[14] may be (<70% bootstrap value) a strain from Egypt in 2008 (RVA/Human-wt/EGY/EGY3399/2008/G6P[14]), as these strains share 70% nucleotide sequence identity. Phylogenetic analysis showed that the strains RVA/Human-wt/GR/20200509/2885/2020/G8P[14], RVA/Human-wt/GR/20210605/2974/2021/G6P[14], and RVA/Human-wt/GR/20210521/3005/2021/G8P[14] formed a subcluster with the MW727449.1 red fox strain RVA/Fox-wt/HRV/L157-VS/2018/G8P[14] from Croatia in 2018, with a bootstrap value < 70% and 99% nucleotide identity. This subcluster belongs to a cluster with bootstrap probability of < 70% that includes the strains RVA/Human-wt/HUN/182-02/2002/G8P[14], RVA/Human-wt/GR/20120422/19–38/2012/G6P[14], and RVA/Human-wt/GR/20120417/1100/2012/G8P[14], together with the fox strain (99% nucleotide sequence identity).

The strain RVA/Human-wt/GR/20200422/2887/2020/G8P[14] was found to be distantly related to the other samples from our study and showed less than 70% relation to the human strain RVA/Human-wt/VNM/NT0082/2007/G10P[14] from Vietnam in 2007, with 95% nucleotide sequence identity. The strain RVA/Human-wt/GR/20210516/3096/2021/G6P[14] was also found to be distantly related (< 70%) to other European strains, with 95–96% nucleotide sequence identity.

Discussion

In this study, we investigated the epidemiology of unusual RVA strains detected in 62 hospitalized children with rotavirus AGE from 2007 to 2021 in Greece and performed a genetic and phylogenetic analysis of P[14] strains. In recent years, an increase in the circulation of unusual RVA strains has been detected. Multiple variants were detected in the antigenic epitopes of the VP8* region, but their significance is unknown. A distant evolutionary relationship was found between Greek human P[14] RVA strains and a Croatian fox P[14] RVA strain, which suggests a possible zoonotic origin of P[14] and interspecies transmission from a wild animal to humans.

Data obtained before the implementation of the RVA vaccine demonstrate that the genotypes P[6], P[9], and P[11] circulated at a significantly higher rate in America, Africa, and Asia than in Europe and Australia, while G8 mainly circulated in Africa [22]. The implementation of vaccines has an impact on the genotype distribution, resulting in the detection of a greater variety of genotypes around the world, with an increase in G9 and G12 as well as in unusual genotypes [23, 24]. Changes in genotype distribution were also observed during the COVID-19 pandemic. In Australia, from 2020 to 2021, a remarkable increase in G8P[8] from 1–87.5% was reported, with a peak in autumn, while the prevalence of G8P[14] decreased from 7.1% to < 1% [25, 26]. In a longitudinal study from 2014 to 2020 in Japan, the frequency of G8P[8] increased from 34% in 2017–2018 to 98% in 2018–2019, peaking in the spring [27]. In Europe, the genotypes G8P[8] and G8P[14] were detected at a prevalence of > 1%, in contrast to previous years [28]. Similarly, in our study, in the period 2019–2021, many different unusual genotypes were detected at > 1% prevalence, including P[9] and P[10] in 2019, P[9], P[14], G8, and G10 in 2020, and P[9], P[14], and G6 in 2021, with a seasonal peak in the spring.

P[14] is an unusual genotype, and its frequency differs in different countries. In our 15-year study, its frequency was < 1%. Similar to what has been observed in other countries, P[14] was mostly found together with genotype G8 in our study. In Venezuela, before the implementation of an RVA vaccine, the G8P[14] was the sixth most common RVA genotype, but after the start of vaccination, G8P[14] (2.4%) was the third most common RVA genotype detected, after G2P[4] and G1P[8] [29, 30]. In Japan, only one out of 247 RVA-positive samples had the G8P[14] combination. This sample was detected in the winter of 2014 [31], in contrast to our P[14] samples, which were mostly detected in the spring.

In this study, in addition to G8, the P[14] genotype was also combined with G6 and G4. Genotypes G6 and G8, have been detected sporadically (< 1%) in humans in previous studies, suggesting zoonotic origin [32,33,34], primarily from bovines [35, 36]. These genotypes are more common in countries that do not follow strict hygiene measures and people come into contact very often with farm animals as well as wildlife [37].

Although G8 and G6 are common in bovines [22], G8 is also found in oysters and shellfish, although it does not infect them [38]. In Bangladesh, the predominant genotype (> 94%) among 200 diarrheic calves was G6P[11] [39], while during 2017–2019 in India, G6P[14] and G8P[14] were the most common RVA genotypes in bovines [40]. In Brazil, in 2010–2016, only 3% of children < 3 years old with AGE were infected with G8P[4] [41], while in West Africa, in 2010, an infant with diarrhea was infected with RVA G6P[14] [42]. In a study in Pakistan in 2015 that included 180 samples from children < 5 years old with AGE, G8 was among the most common RVA strains in infants [43], but in a study in Iran in the period of 2017–2019, G8, G6, and P[14] were not detected in any of 130 children < 15 years old with AGE [44]. Also, G8 strains were reported by EuroRotaNet as significant emerging strains in the UK during 2008–2010 and 2018–2019 (EuroRotaNet, annual report 2019).

Recently, a new combination, G9P[14], was detected in Egypt in a 6-month-old child with AGE [45]. The sequence of P[14] in this strain was very similar to the old sequence EF554107 (95.45%) from Hungary [45]. This probably indicates that P[14] has been conserved over time.

Previous phylogenetic and genetic analysis of RVA strains from feces of humans and animals with gastroenteritis has suggested that P[14] has a zoonotic origin, most likely from bovines. A study from Japan showed that P[14] sequences from humans were similar to sequences present in bovine strains [31]. In this study, we found a distant relationship between human P[14] RVA and fox P[14] RVA, with a bootstrap value < 70%. This finding suggests the interspecies transmission between wild animals and humans.

So far, there is only one study from Croatia, where foxes live near urban areas, that refers to RVA infection in fox feces. It was shown that one red fox (Vulpes vulpes) was infected with a G8P[14] RVA strain in 2018 [46]. Therefore, wild animals such as 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. Studies from various countries have demonstrated the susceptibility of P[14] to reassortment events. For example, NGS of two G8P[14] strains isolated from children with AGE in Italy in 2012 provided evidence of reassortment events between RVA strains from humans and sheep [47]. In a Slovenian study, an RVA strain isolated from a 1-year-old child with severe AGE in 2009 was genotyped using NGS, revealing a likely zoonotic origin of that strain (G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3) [48]. In another study in Hungary, genome analysis of a G8P[14] isolate from a 4-year-old child with AGE in 2001 using NGS revealed that more than one reassortment event had occurred in this strain (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) [49]. In Honduras, NGS of an RVA G10P[14] strain isolated from a 30-month-old child with severe AGE also revealed multiple reassortment events, suggesting a zoonotic origin of this strain, and potential reservoirs were identified [50]. All of these strains were pathogenic to humans, but the specific features of the virus that lead to severe AGE remain unknown. Larger studies or meta-analyses need to be conducted to address this question.

It is known that the viral proteins VP8* (derived from VP4) and VP7 play a significant role in attachment of the virus to cellular receptors containing sialic acid (SA), histo-blood group antigens (HBGAs), or cell surface components such as sialoglycans [11, 51, 52]. During endocytosis of the RVA virion, VP4 undergoes structural changes that expose hydrophobic sites of the protein [53]. VP4 contains three highly conserved trypsin cleavage sites at arginine residues 231, 241, and 247, where it is divided into two polypeptides, VP5* and VP8*, the exact length of which depends on which site is used for cleavage [54]. Cleavage of VP4 activates the infectivity of the virus [55]. In all of our samples, these regions were found to be conserved. However, we found many amino acid variants near significant sites or within antigenic epitopes. In contrast to our results, analysis of the antigenic epitopes of two G8P[14] strains in Italy showed that the sequences were conserved [47]. Further studies using mutagenesis of these strains to investigate the role of specific variants in the pathophysiology and severity of disease should be conducted.

A limitation of our study is that we did not determine the complete genotype constellations of the P[14] strains in order to identify possible reassortment events and determine their evolutionary history based on complete genome sequences. This is the first report of unusual P[14] RVA strains detected in Greece focusing on phylogenetic analysis of a part of the VP4 gene, which suggests their potential zoonotic origin. Previous studies have shown the genotype distribution of common and less common genotypes circulating in Greek children [19, 24, 56, 57]. Continuous surveillance of the distribution of RVA genotypes and their evolution is crucial for a better understanding of this virus, the disease that it causes, and the potential need to develop new RVA vaccines.

Conclusions

In this longitudinal study, the genotype distribution of unusual G and P rotavirus strains was investigated. The long-term implementation of the RVA vaccine as well as the COVID-19 pandemic seem to have a significant impact on the epidemiology of the virus and its genotype distribution, resulting in an increase in the circulation of unusual strains. Constant surveillance of RVA genotypes is necessary for assessing the need for the development of new vaccines and estimating the breadth of coverage of circulating RVA genotypes by existing vaccines.