Genotyping of VP6 and NSP4 genes and molecular characterization of NSP4 gene of unusual Rotavirus group A isolated from children with acute gastroenteritis

doi:10.21203/rs.3.rs-3609731/v1

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Genotyping of VP6 and NSP4 genes and molecular characterization of NSP4 gene of unusual Rotavirus group A isolated from children with acute gastroenteritis

https://doi.org/10.21203/rs.3.rs-3609731/v1

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Group A Rotavirus (RVA), which causes acute gastroenteritis (AGE) in children worldwide, is categorized mainly based on VP7 (genotype G) and VP4 (genotype P) genes. Genotypes that circulate at < 1% are considered unusual. Important genes are also VP6 (genotype I) and NSP4 (genotype E). VP6 establishes the group and affects immunogenicity, while NSP4, as enterotoxin, is responsible for the clinical symptoms. Aim of this study was to genotype the VP6 and NSP4 genes and molecularly characterize the NSP4 gene of unusual RVA. Unusual RVA strains extracted from fecal samples of children ≤ 16 years with AGE, were genotyped in VP6 and NSP4 genes with Sanger sequencing. Phylogenetics was performed using MEGA 11. In a 15-year period (2007–2021), 54.8% (34/62) of unusual RVA were successfully I and E genotyped. Three different I and E genotypes were identified; I2 (73.5%, 25/34) and E2 (35.3%, 12/34) were the commonest. E3 genotype was detected from 2017 onwards. The uncommon combination of I2-E3 was found in 26.5%(9/34) of the strains and G3-P[9]-I2-E3 was the most frequent G-P-I-E combination (20.6%,7/34). Statistical analysis showed that children infected with E2 strains had a higher relative frequency of dehydration(50%) compared to those with E3 genotype(p = 0.019). Multiple substitutions were detected in NSP4, but their functional effect remains unknown. The results indicate the genetic diversity of RVA strains. Continuous surveillance of the RVA based on the whole genome will provide a better knowledge of its evolution.

Rotavirus

acute gastroenteritis

children

NSP4

VP6

genotyping

Group A Rotavirus (RVA) is one of the most common etiological agents of acute gastroenteritis (AGE) in infants and young children, especially in developing countries. Children with RVA AGE can present severe dehydration that can even lead to death if left untreated. RVA is responsible for more than 100,000 deaths each year worldwide [1].

RVA is a non-enveloped, icosahedral, double stranded RNA virus (dsRNA) and is a member of the Reoviridae family. Its genome consists of 11 linear dsRNA segments which encode six structural viral proteins (VP1-VP4, VP6 and VP7) and six non-structural viral proteins (NSP1-NSP6) [2].

The viral particles consist of a triple layered capsid. The outer capsid consists of the glycoprotein VP7 and the spike protease-sensitive protein VP4. The middle layer consists of VP6 and the core layer comprises of the VP2 which encapsulates genomic RNA and viral replication components [2]. The abundant VP6 protein is commonly used for the detection and classification of rotaviruses. Currently, nine rotavirus species (A-D and F-J) have been recognized by the International Committee on Taxonomy of Viruses (ICTV); however, two additional putative rotavirus species K and L have also been proposed [3]. Only species A, B, C and H can infect humans including animal-human transmissions [4].

RVAs are further classified based on the outer layer proteins VP7 and VP4 in G and P genotypes, respectively. Although many different G and P types have been identified so far the most common circulating genotypes are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] [5, 6]. Genotyping can also be applied in the whole virus genome, Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, with “x” indicating the number of the corresponding genotype, which represents the genotypes of the genes VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 [7].

NSP4, except for the full length protein, encodes a toxic-peptide (114–135 amino acids) and both act as enterotoxins that can stimulate Ca²⁺ release from the endoplasmic reticulum into the cytoplasm [8, 9]. NSP4 enterotoxin activity aggravates the symptoms of gastroenteritis and especially diarrhoea and vomiting [10, 11].

Since 2006, several RVA vaccines have been released worldwide. The most widely used are the two-dose monovalent vaccine Rotarix (GlaxoSmithKline Biologicals, Belgium) and the three-dose pentavalent vaccine RotaTeq (Merck, United States), which cover the most common G and P genotypes [12, 13]. After their release, notable changes in genotype distribution have been described worldwide, such as the increase in unusual G and P genotypes [2, 14]. The aim of this study was the genotyping of VP6 and NSP4 genes as well as the molecular and phylogenetic characterization of NSP4 gene of previously described [15] unusual G and P RVA strains isolated from children ≤ 16 years with AGE.

Study design

This is a retrospective study involving RVA positive fecal samples with previously described unusual G (G6, G8, G10) and/or P (P[6], P[9], P[10], P[11], P[14]) genotypes collected from children ≤ 16 years hospitalized with AGE [15]. In the present study, these strains were further genotyped in VP6 (I genotype) and NSP4 (E genotype) genes.

Demographic and epidemiological data such as age, gender, residence, and RVA vaccination status were also collected from the children infected with an unusual RVA genotype. Clinical symptoms (diarrhoea, vomiting, fever and dehydration) and laboratory data (measurements of potassium (K⁺), sodium (Na⁺), calcium (Ca²⁺), Chlorine (Cl^-), C-reactive protein (CRP), urea, creatinine, white blood cells (WBC), polymorphonuclear leukocytes and lymphocytes) were also recorded.

The Scientific and Bioethics Committee of “Aghia Sophia” Children’s Hospital approved this study (No 6261).

Reverse transcription and amplification of VP6 and NSP4 genes

Nucleic acid extraction and reverse transcription (RT) were performed as previously described [15]. PCR amplification was performed using GoTaq DNA polymerase (Promega; Madison, WIS, USA) and primers F: 5’-GAC GGV GCR ACT ACA TGG T-3’ and R: 5’-GTC CAA TTC ATN CCT GGT G-3’ for the VP6 gene and F: 5’-GGC TTT TAA AAG TTC TGT TCC GAG-3’ and R: 5’-GTC ACA YTA AGA CCR TTC CTT CCA T-3’ for NSP4 gene [16, 17]. The PCR amplification was carried out with an initial denaturation at 94οC for 2 minutes (min), followed by 40 cycles of denaturation for 1 min at 94^οC, annealing for 1 min at 55^οC for the VP6 gene and 48^οC for the NSP4 gene, extension for 1 min at 72^οC and final extension for 10 min at 72^οC. The amplification products were analyzed by 2% agarose gel electrophoresis using a 50bp DNA ladder (N3236S; New England Biolabs, Massachusetts, USA) and ethidium bromide staining. If any of these two products was not produced, the genotype was characterized as identified (UD).

Sequencing and phylogenetic analysis

The I and E genotypes was determined by performing Sanger sequencing with the BigDye Terminator v3.1 cycle sequencer kit on an Applied Biosystems 3500 genetic analyzer (Applied Biosystems, Waltham, MA, USA) and using the BLAST bioinformatic tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The NSP4 sequences were compared to reference strains from the Wa, DS-1, and AU-1 constellations depending on the E genotype to detect substitutions. Reference strains AF093199.1 (Wa), EF672582.1 (DS-1) and D89873.1 (AU-1) were used for the molecular characterization of the NSP4 sequence.

Phylogenetic evolutionary analysis was performed on NSP4 gene using the MEGA 11 software (Molecular Evolutionary Genetics Analysis; www.megasoftware.net). Multiple sequence alignment was performed using MUSCLE software (Multiple Sequence Comparison by Log-Expectation). The nucleotide substitution model was selected based on the BIC (Bayesian Information Criterion) scores using MEGA11. The model used in this study was the Tamura 3-parameter (T92) and the rate variation model, which allows some sites to be evolutionarily invariant (+ I). The evolutionary tree was constructed using the Maximum Likelihood method and bootstrap resampling with 1000 replicates.

Statistical analysis

Data statistical analysis was carried out using SPSS software (IBM Statistical Package for Social Sciences for Windows, Version 25.0. Armonk, NY: IBM Corp). p-value ≤ 0.05 was considered statistically significant. A Pearson’s chi-square test (χ² test) was applied to determine the differences for the variables that met the criteria of their application. For variables that did not meet the criteria of the χ² test or both variables had two categories (2x2 double entry matrix), Fisher’s exact test was used.

Nucleotide sequence accession numbers

The NSP4 nucleotide sequences of this study were deposited in GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) with accession numbers OM281957-59, OM287400, OM303085, OM303088, OM333185, OM333186, OM323986, OM461377, OM461378, OM972707-OM972710, ON004913, ON009342, ON156796, ON156797, ON185611-18, ON206978, ON206980-83, ON971933, ON971934 for the VP6 gene and OM281953, OM281956, OM283121-26, OM287398, OM287399, OM362404, OM948988-91, ON004914, ON156785-93, ON564370, ON564371, ON971935 for the NSP4 gene (Suppl Table 1).

I and E genotyping and genetic linkage with G and P genotypes

From 2007 to 2021, 54.8% (34/62) of the unusual RVA strains were successfully I and E genotyped, and they consisted of 5.9% (2/34) unusual G (G8 and G10), 64.7% (22/34) unusual P (P[6], P[9], P[10] and P[11]) and 29.4% (10/34) unusual G and P (G6P[9], G6P[14] and G8P[14]).

Three different I and E genotypes were identified: I1 (7/34, 20.6%), I2 (25/34, 73.5%), I3 (2/34, 5.9%) and E1 (5/34, 14.7%), E2 (12/34, 35.3%), E3 (11/34, 32.4%). The most common genotypes were I2 and E2. The E3 genotype was first detected in samples in 2017 and was the second most common E genotype (Fig. 1). E3 was detected in strains with P[9] genotype combined with G3 (n = 8), G4 (n = 1), G6 (n = 1), and G9 (n = 1) genotypes (Table 1).

Table 1

Unusual G-P-I-E genotypes of Group A Rotaviruses circulating in Greece in a 15-year period (2007–2021).
Genotypes (Gx-Px-Ix-Ex)			%
Strains with G unusual genotype	G8-P[8]-I1-EUD	1	2.94
Strains with G unusual genotype	G10-PUD-I2-E2	1	2.94
Strains with P unusual genotype	G3-P[9]-I2-E3	7	20.59
	G3-P[9]-I2-E2	2	5.89
	G9-P[10]-I1-E1	2	5.89
	G2-P[6]-I2-EUD	1	2.94
	G3-P[9]-I3-E3	1	2.94
	G4-P[6]-I2-E2	1	2.94
	G4-P[6]-I1-E1	1	2.94
	G4-P[9]-I1-E1	1	2.94
	G4-P[9]-I3-E3	1	2.94
	G9-P[9]-I2-E2	1	2.94
	G9-P[9]-I2-E3	1	2.94
	G12P[6]-I1-E1	1	2.94
	G12P[6]-I1-EUD	1	2.94
	G12P[11]-I1-E2	1	2.94
Strains with G and P unusual genotype	G8-P[14]-I2-E2	4	11.76
	G6-P[9]-I2-EUD	2	5.89
	G6-P[9]-I2-E2	1	2.94
	G6-P[9]-I2-E3	1	2.94
	G6-P[14]-I2-E2	1	2.94
	G8-P[14]-I2-EUD	1	2.94
Total		34	100
Abbreviation: EUD = unidentified E genotype.

Six (6/34, 17.6%) samples were not successfully genotyped in NSP4 gene and they were characterized as EUD (unidentified E genotype). These RVA strains were the following: G2-P[6]-I2-EUD (n = 1), G6-P[9]-I2-EUD (n = 2), G8-P[8]-I1-EUD (n = 1), G8-P[14]-I2-EUD (n = 1), and G12-P[6]-I1-EUD (n = 1) (Table 1).

The most frequent combinations of G-P-I-E were G3-P[9]-I2-E3 and G8-P[14]-I2-E2 accounting for 20.6% (7/34) and 11.8% (4/34) of the samples, respectively (Table 1).

Association of I and E genotypes with patient characteristics

Statistical analysis of demographic, clinical, and laboratory data from children depending on RVA I genotype showed no significant correlation. The corresponding analysis with E genotypes showed that children infected with E2 RVA strains had a higher relative frequency of dehydration (6/12, 50%) compared to those with the E3 genotype (0/9, 0%) (p = 0.019).

Molecular characterization and phylogenetic analysis of NSP4

Molecular characterization was performed οn the whole NSP4 gene. Through this comparison, 13 homozygous missense substitutions were found in strains carrying the E1 genotype, 21 homozygous and two heterozygous missense substitutions in strains carrying the E2 genotype and 16 homozygous and one heterozygous missense substitution in strains carrying the E3 genotype (Fig. 2). Most of these substitutions (n = 23) were located in the VP4 binding region (aa 112–148).

The NSP4 gene sequences were compared with the 100 most similar strains using BLAST, and eight possibly novel substitutions were identified. These novel substitutions were the D140N in one E1 strain, the L25I (n = 1), T78A (n = 1) and D140N (n = 2) in four E2 strains and the D19G (n = 1), I24V (n = 1), V102I (n = 1), K141R (n = 2) and T155M (n = 2) in six E3 strains. Four of these substitutions were located within significant domains of NSP4. Specifically, D19G and T78A were in the conserved hydrophobic domains 1 and 3 (H1, H3), respectively, and the D140N and K141R were located in VP4 binding domain.

In the toxic peptide region, three already known homozygous substitutions were detected. The H131Y was found in 1/5 (20.0%) E1 strain, in 4/12 E2 strains (33.3%) and in 1/11 (9.1%) E3 strain. The M133V was found in 7/11 (63.6%) E3 strains and the M135V was detected in 1/12 (8.3%) E2 strain (Fig. 2).

Phylogenetic analysis of the NSP4 gene in 28 unusual RVA strains revealed three distinct groups corresponding to E1, E2, and E3 genotypes with 100% reliability. Among the unusual RVA strains carrying the E2 genotype, three distinct clades (E2-A, E2-B and E2-C) were identified (Fig. 3). The division of the E2-A clade from E2-B and E2-C is based on four synonymous substitutions (L21L/c.63A > G, I56I/c.168A > T, L116L/c.346C > T, V124V/c.372A > T). The E2-A clade differentiated from the E2-B clade due to one missense (A45T/c.133G > A) and additional five synonymous (N18N/c.54T > C, Q109Q/c.327A > G, L110/c.330A > G, I130I/c.390C > T, S138S/c.414G > A) substitutions and from the E2-C clade due to one missense (G140D/c.419G > A) and another five synonymous (P34P/c.102C > T, E125E/c.375G > A, I130I/c.390A > T, P168P/c.504G > A) substitutions. The E2-B clade differed and separated from the E2-C clade due to two missense (A45T/c.133G > A, G140D/c.419G > A) and nine synonymous substitutions.

Unusual strains carrying the E1 and E3 genotypes were also divided into 3 (E1-A, E1-B, E1-C) and 2 (E3-A, E3-B) distinct clades, respectively (Fig. 3). Separation between the E1-A and E1-B strains occurred due to three missense (I141V/c.421A > G, T145S/c.433A > T, I169S/c.505_506AT > TC) and 22 synonymous substitutions. The E1-C clade differed from both E1-A and E1-B clades due to two missense (I76V/c.226A > G, S161N/c.482G > A) and three synonymous (K3K/c.9G > A, L82L/c.244_246TTG > CTA, P138P/c.414A > G) substitutions. Furthermore, the E1-C clade differed from the E1-A clade in three missense (V141T/c.421_422GT > AC, S145T/c.433T > A, S169I/c.505_506TC > AT) and 21 synonymous substitutions and from the E1-B clade in one missense (I141T/c.422T > C) and seven synonymous substitutions. The division among the E3 cluster appeared due to six missense (I51V/c.151A > G, R59K/c.176G > A, R141K/c.422G > A, F148I/c.442T > A, R151K/c.452G > A, Q152H/c.456A > C) and 25 synonymous substitutions (Fig. 3).

There are limited studies that investigate the molecular characterization of VP6 and NSP4 genes in human RVA strains worldwide as the interest has mainly focused on G and P distribution. This 15-year study focusses on the genotyping of VP6 and NSP4 genes and molecular characterization of the NSP4 gene of unusual G and P RVA strains isolated from children hospitalized with AGE.

Genotyping revealed three different I (I1, I2, I3) and E (E1, E2, E3) genotypes in unusual RVA strains, I2 and E2 being the most common. According to the Rotavirus Classification Working Group, 32 I and E genotypes are known so far, with I1, I2 and E1, E2 being the most commonly detected genotypes among humans [8, 20–22]. I1-E1 are strongly associated with G1/G3/G4/G5/G9-P[8] and follow the Wa-like genotype constellation, I2-E2 are associated with G2-P[4] typical of the DS-1 like genotype constellation and I3-E3 are associated with G3-P[9] typical of the AU-1 like constellation [21, 23].

Similarly, in a 10-year study (1996–2006) conducted in Brazil, which included both common and unusual strains, they found the same three I and E genotypes [23]. ] In their study the most prevalent I and E genotypes were I1 (82.7%) and E1 (81.5%), respectively. However, among strains with an unusual G (G6, G8, G10) and/or P (P[6], P[9], P[10], P[11], P[14]) genotype, I2 and E2 were the most prevalent I and E genotype (n = 7/13) [23], as in the present study. In other epidemiological studies such as a 4-year study conducted in the Democratic Republic of Congo, although the number of unusual G and/or P strains recorded was substantial, only two I (I1, I2) and E (E1, E2) genotypes were recorded [24].

In this study, I3 and E3 were detected in 2019 and 2017 onwards, respectively. Strains carrying the E3 genotype showed a significant increase between 2019–2021, during the COVID-19 pandemic period and they were mostly found in combination with G3-P[9]-I2 (n = 5/21, 23.8%). G3-P[9]-I2-E3 was the most prevalent G-P-I-E genotype combination throughout this study (n = 7/34, 20.6%). The increase in E3 was observed in the same period with the increase of P[9] strains in Greece, as recorded by Tatsi et al [15]. The first record of the G3-P[9]-I2-E3 genotype in humans was in 2012 in Korea, where it was isolated from a 9 year old female [25]. However, a similar strain (G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3) was recently identified in 2021 in Thailand, and was originated from a feline with diarrhoea [26].

The rare combination of G3-P[9]-I2-E3 that was detected in this study is possibly derived from a reassortment event, but further investigation should be performed. Reassortment is common among RVs and is a crucial mechanism for the evolution of the virus. Molecular characterization of multiple RVA genes is important, as it may contribute to detect strains that do not fit into any of the major constellations (Wa, DS-1 and AU-1) and are probably products of reassortment events. Furthermore, this finding supports that VP6 and NSP4 can segregate independently, contradicting a study in 2003 that reported a genetic linkage among these two proteins in common, unusual and reassortant human strains [21]. Similarly, to our observation, many studies reported such reassortment events at VP6 and NSP4, but at a lower rate. In an 11-year study (1996–2006) in Brazil, the I1-E2 unusual I-E genotype combination was found in 1.2% of circulating strains [23]. The combinations I2-E1 and I1-E2 were detected in 15.4% of RVA strains in India during 1990–2000[27] and in 6.5% in Iran during 2021–2022 [28].

NSP4 is an essential protein for virus morphogenesis and pathogenesis. In the present study, nine possibly novel substitutions were found in the NSP4 gene. Most substitutions were detected in VP4-binding domain which also contains the toxic peptide and the interspecies variable domain (ISVD). According to other studies characterizing the nucleotide sequence of the NSP4 gene, the ISVD region shows great heterogenicity and the amino acid vary according to genotype [17, 29–32]. Limited functional studies exist and therefore the effects of these variants on the functionality and immunogenicity of the corresponding protein remain unknown.

Of interest are the substitutions in amino acid 131 in the region of the toxic peptide, in which the majority of the strains of this study carried the H131 and E2 strains mainly carried Y131. Ball et al. conducted functional study for this amino acid on infant mice and they found that substitutions in amino acid 131 has an effect on the enterotoxin properties of NSP4.[33] Specifically, they reported that the Y131K substitutions resulted in the absence of diarrhoea. Studies from Brazil between 1990–2000 and 1987–2003 have reported that Y131 was detected only in E2 strains, while E1 strains had H131, and there was no data regarding E3 strains.[34, 35] Srivastava et al. showed that patients infected with a strain carrying Y131 experienced more severe diarrhoea [29]. Even though the severity of symptoms was not evaluated in the present study, statistical analysis showed that children infected with an unusual strain carrying the E2 genotype had a higher chance to exhibit dehydration, which may indicate more severe diarrhoea. This result may also be related to the fact that Y131 was detected more in E2 strains.

Limitation of the present study included the moderate detection rates of both VP6 and NSP4 genes in RVA-positive fecal samples. However, similar detection rates have also been reported in other studies, possibly due to poor sample storage conditions or the presence of RNases resulting in fragmentation of the viral RNA genome, presence of PCR inhibitors or inability of primers to hybridize [8, 36]. Another limitation of our study was that the analysis was based only in four genes (VP7, VP4, VP6 and NSP4) and not in the complete genotype constellation, which would provide more information about the genetic evolution of the strains.

This is the first study of VP6 and NSP4 epidemiology and molecular characterization of NSP4 of unusual RVA strains in Greece, in which the unusual I3 and E3 genotypes, the reassortant I2-E3 human strains and many substitutions in significant domains of NSP4 gene were detected. Furthermore, a significant clinical association between dehydration and E2 genotype was described.

Continuous surveillance of the distribution of RVA genotypes based on the whole genome, the molecular characterization and their association with epidemiological and clinical data is important for the better knowledge of the virus’ evolution, the disease prognosis and upgrading RVA vaccines.

In this study, the genotype distribution of the VP6 and NSP4 gene in unusual rotavirus strains was described. The association between RVA genotype and the severity of the symptoms needs to be further investigated. The application of next generation sequencing to investigate genotypic combinations in the complete viral genome in combination with phylogenetic analysis will probably provide answers to the origin and evolutionary relationship of these strains.

Competing interest: All authors declare that they have no competing interests regarding the present study.

Ethical approval: The study protocol was approved by the scientific and bioethics committee of “Aghia Sophia” Children’s Hospital (No. 6261) and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

Data availability: All relevant data are within the paper.

Author contributions E.B.T., D.M.K., and V.S. contributed to the conception of the study. C.D., E.B.T., D.M.K., F.F. and E.E.V. collected samples and demographic data. C.D., D.M.K., and E.B.T carried out the experiments. C.D. and E.B.T. analyzed the data. C.D. performed the phylogenetic analysis and wrote the initial manuscript. A.M., V.S. and E.B.T. supervised the study. All authors reviewed and approved the final manuscript.

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Genotyping of VP6 and NSP4 genes and molecular characterization of NSP4 gene of unusual Rotavirus group A isolated from children with acute gastroenteritis

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

Study design

Reverse transcription and amplification of VP6 and NSP4 genes

Sequencing and phylogenetic analysis

Statistical analysis

Nucleotide sequence accession numbers

RESULTS

I and E genotyping and genetic linkage with G and P genotypes

Association of I and E genotypes with patient characteristics

Molecular characterization and phylogenetic analysis of NSP4

DISCUSSION

CONCLUSIONS

Declarations

References

Additional Declarations

Supplementary Files

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