Short communicationGenetic heterogeneity and recombination in type-3 human astroviruses
Introduction
Human astroviruses (HAstVs), genus Mamastrovirus, family Astroviridae, are important etiological agents of gastroenteritis in humans, mostly in young children, elderly people, and immunocompromised patients (Mendez and Arias, 2013). HAstVs have a single stranded positive-sense RNA of about 6.8 kb in length that contains three overlapping open reading frames (ORFs). ORF1a, ORF1b, and ORF2 encode the serine protease, the RNA-dependent RNA polymerase, and the capsid protein precursor, respectively. The capsid protein precursor can be divided into a highly conserved N-terminal domain (amino acids [aa] 1–424), a hypervariable (HVR) domain (aa 425–688), and a highly acidic C-terminal domain (Wang et al., 2001). The HVR domain of HAstV is believed to form the capsid spike and to control binding to cell receptors as neutralizing epitopes have been mapped inside this capsid portion (Dong et al., 2011).
Studies based on immune electron microscopy, immunofluorescence, ELISA and plaque neutralization assays have revealed that HAstVs are antigenically heterogeneous and that they can be classified into eight serotypes (HAstV-1 to HAstV-8). (Bosch et al., 2014, Mendez and Arias, 2013, Kurtz and Lee, 1984). Sequence analysis of short fragments at either the 5′ or 3′ end of ORF2 (regions C and D) and RT-PCR genotyping protocols with type-specific primers have been used for genetic characterization of HAstV-1 to -8 (Belliot et al., 1997, Noel et al., 1995). HAstV-1 appears to be the predominant circulating serotype worldwide followed by types 2–5 and occasionally by type-8, depending on the region. HAstV-6 and -7 are rarely detected (De Grazia et al., 2012, Gabbay et al., 2007, Guix et al., 2002, Liu et al., 2008, Medici et al., 2012, Mendez-Toss et al., 2004, Mustafa et al., 2000).
Also, sequence analysis of the small diagnostic regions located on ORF2 has revealed discrete sequence variation within some HAstV types, revealing intra-typic genetic lineages. Type-1 HAstV has been classified into four lineages (HAstV-1a to -1d), type-2 into four (HAstV-2a to -2d), type-3 into two (HAstV-3a and -3b) and type-4 into three (HAstV-4a to -4c) (De Grazia et al., 2012, Gabbay et al., 2007, Guix et al., 2002, Martella et al., 2013). Comparison of the full-length ORF2 has confirmed the classification of HAstVs into discrete lineages. Cutoff values of 5–6.5% nucleotides (nt) identity in the full-length ORF2 have been calculated among the various lineages. These analyses have also revealed several examples of intra-typic recombination within this genomic region (Martella et al., 2014). Also, examples of recombination in, or close to the ORF1b/ORF2 junction have been described, suggesting that this genomic region may be a preferential site for RNA cross-over (Babkin et al., 2014, De Grazia et al., 2013, Gabbay et al., 2007, Martella et al., 2013, Walter et al., 2001).
Surveillance for HAstV in Parma, Northern Italy, has been carried out continuously since 2008 in children aged <5 years hospitalized with acute gastroenteritis at the Maternal–Infantile Department of the University Hospital of Parma. All the HAstV strains identified between 2008 and 2013 were systematically genotyped in the diagnostic region C (nt 4571–4918 of accession No. L13745) (De Grazia et al., 2013, Martella et al., 2013, Medici et al., 2012). In this 6-years period, the overall prevalence of HAstV infection was 1.34% (32 out of 2383 children with gastroenteritis), ranging from 3.87% in 2008 to 0.49% in 2011. Five different genotypes were found to circulate in this time span, with type-1 HAstVs being predominant (23 cases, 71.88%), followed by type-2 (4 cases, 12.5%) and type-4 (3 cases, 9.38%), type-3 (1 case, 3.12%) and type-5 (1 case, 3.12%). The type-1 HAstVs were sub-typed as lineage 1a (16 cases, 69.56%), 1b (1 case, 4.35%) and 1d (6 cases, 26.09%). All the type-2 HAstVs were characterized as lineage 2d, and all the type-4 HAstVs as lineage 4c, while the type-5 HAstV strain was sub-typed as 5c. Based on the small sequence generated in ORF2 region C, the single HAstV-3 strain, PR1365/2012/ITA, detected in April 2012, segregated within a novel, yet unrecognized, type-3 lineage, along with similar strains detected globally. In order to investigate better the genetic signature of this strain, a 3.2 kb portion at the 3′ end of the genome was sequenced and compared with HAstV-3 strains retrieved from the databases.
Section snippets
Materials and methods
A 3′ RACE-PCR protocol was used to generate a 3.2 kb amplicon encompassing the 3′ end of ORF1b, the full-length ORF2, the 3′ untranslated region (UTR) through the poly-A tail (Wang et al., 2005). Briefly, cDNA was synthesized by SuperScript III First-Strand cDNA synthesis kit (Invitrogen Ltd, Paisley, UK) with primer VN3T20 (5′-GAGTGACCGCGGCCGCT20-3′). PCR was performed with TaKaRa La Taq polymerase (TaKaRa Bio Europe SAS, Saint-Germain en-Laye, France) with forward primer panAstVFor1
Results and discussion
The 42 HAstV sequences retrieved from the databases encompassed type-3 strains detected worldwide over a nearly 3-decade period. Three distinct lineages were clearly resolved by phylogenetic inference in either the full-length ORF2 or the region C of ORF2, and these clusters were statistically supported (Fig. 1). Considering the whole data set, 30 (71.42%) sequences were characterized as lineage 3a, 3 (7.14%) as lineage 3b and 9 (21.42%) as the novel, yet unrecognized lineage, tentatively
Conclusions
Although several sequences of the HAstV lineage 3c were already available in the databases, with the oldest strains dating back to the late 1990s, this different type-3 lineage had not been recognized thus far, and none of these type-3 viruses was characterized more in detail. Phylogenetic analysis based on the small sequences of the diagnostic region C of ORF2 was consistent with the analysis based on the full-length ORF2, confirming that region C is a good proxy for prediction of HAstV types
Acknowledgments
This study was partly supported by the Grant “Ricerca Scientifica FIL 2012,” University of Parma, Italy (Fondi di Ateneo 2012). The authors have no conflicting interests to declare.
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