Zoonotic transmission of reassortant porcine G4P[6] rotaviruses in Hungarian pediatric patients identified sporadically over a 15 year period

Highlights

• The complete genotype configurations of 8 Hungarian G4P[6] rotaviruses were determined.

• Porcine and porcine derived human rotavirus genes were identified in the genomes of all 8 human G4P[6] strains.

• These strains emerged probably through independent interspecies transmission events.

Abstract

Genotype G4P[6] Rotavirus A (RVA) strains collected from children admitted to hospital with gastroenteritis over a 15 year period in the pre rotavirus vaccine era in Hungary were characterized in this study. Whole genome sequencing and phylogenetic analysis was performed on eight G4P[6] RVA strains. All these RVA strains shared a fairly conservative genomic configuration (G4-P[6]-I1/I5-R1-C1-M1-A1/A8-N1-T1/T7-E1-H1) and showed striking similarities to porcine and porcine-derived human RVA strains collected worldwide, although genetic relatedness to some common human RVA strains was also seen. The resolution of phylogenetic relationship between porcine and human RVA genes was occasionally low, making the evaluation of host species origin of individual genes sometimes difficult. Yet the whole genome constellations and overall phylogenetic analyses indicated that these eight Hungarian G4P[6] RVA strains may have originated by independent zoonotic transmission, probably from pigs. Future surveillance studies of human and animal RVA should go parallel to enable the distinction between direct interspecies transmission events and those that are coupled with reassortment of cognate genes.

Introduction

Group A rotavirus (Rotavirus A, RVA) is the single most important cause of childhood diarrhea responsible for an estimated 450,000 deaths each year mostly in developing countries (Tate et al., 2012). RVAs in humans are highly diverse and this diversity is generated primarily by accumulation of point mutations, reassortment of cognate genes, and spillover of animal strains (Matthijnssens et al., 2009a). Historically, RVA strains have been classified into serotypes and genotypes based on the neutralization antigens permitting the utilization of a dual nomenclature where G and P types define VP7 and VP4 specificities, respectively. Thus far 27 G and 37 P types have been identified; of these, surveillance studies performed over the last 30 years globally have identified at least 12 G and 15 P types in humans (Gentsch et al., 2005, Matthijnssens et al., 2009a, Matthijnssens et al., 2011b, Bányai et al., 2012, Trojnar et al., 2013).

More recently, the classification scheme of RVAs has been extended to all 11 genomic segments where particular genotypes are defined by specific cut-off values. In this scheme the whole genome based genotype configuration of the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 encoding genes is described as Gx-Px-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x is replaced with an Arabic number (Matthijnssens et al., 2008). In a recent review 16 I, 9 R, 9 C, 8 M, 16 A, 9 N, 12 T, 14 E, and 11 H genotypes have been listed, and papers published since then described additional new VP4, VP6, NSP4 and NSP2 genotype specificities (Matthijnssens et al., 2011b, Guo et al., 2012, Papp et al., 2012, Trojnar et al., 2013).

Medically important human RVA strains carry G1-G4, G9 or G12 VP7 types and P[4], P[6], or P[8] VP4 types. Combinations of these antigen specificities may be either globally spread (e.g., G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], G12P[8]) or can be prevalent only in particular regions (e.g., P[6] strains with various G type in sub-Saharan Africa). In general, the G1P[8], G3P[8], G4P[8], or G9P[8] antigen combinations are most commonly found in the Wa-like genotype constellation which is characterized by genotype 1 backbone genes (i.e., I1-R1-C1-M1-A1-N1-T1-E1-H1), whereas G2P[4] RVAs are associated primarily with the DS1-like genotype constellation which is specified by genotype 2 backbone genes (i.e., I2-R2-C2-M2-A2-N2-T2-E2-H2) (Matthijnssens et al., 2011b, Matthijnssens and Van Ranst, 2012). Of interest, genotypes of the backbone genes resembling the two major human RVA genotype constellations are prevalent in some animal RVA strains. For example, genotype 1 genes are frequently found in porcine RVA strains, while several genotype 2 genes are common in ruminant RVA strains. This observation suggested that the common human RVA backbone gene configurations may have originated at least partially from animal RVAs (Matthijnssens et al., 2008).

RVA surveillance conducted in Hungary has identified numerous putative RVA strains of animal origin in samples collected over the past two decades (Bányai et al., 2004a, Bányai et al., 2004b, Bányai et al., 2005, Bányai et al., 2009a, Bányai et al., 2010, Matthijnssens et al., 2009b). One example is genotype G4P[6] RVAs which are very rare in pediatric patients in Hungary, representing ⩽ 2% of strains identified in children admitted to hospital due to RVA diarrhea (Bányai et al., 2004a, Bányai et al., 2005, Bányai et al., 2009a, László et al., 2012). Molecular analysis of selected strains have identified unusual P[6] VP4 genes; two novel clusters of the P[6] VP4 gene have been described and subsequent studies indicated that at least one of these novel clusters are closely related to porcine P[6] VP4 gene (Bányai et al., 2004b, Martella et al., 2006, Steyer et al., 2008). In addition, probable porcine derived RVA VP7 genes have been identified in selected Hungarian G4P[6] strains (Bányai et al., 2009a). Mapping the genomic constellation of animal-like RVAs is important to assess whether these strains originate by direct interspecies transmission or by reassortment between homologous and heterologous strains. Also, in the latter case it seems to be of interest to decipher if some genes tend to be selected during adaptation of animal RVAs in the human host. Therefore a more complete genetic characterization of available human G4P[6] RVA strains detected in Hungary was undertaken in this study.