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      First detection of a reassortant G3P[8] rotavirus A strain in Italy: a case report in an 8-year-old child

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          Abstract

          Background

          Acute gastroenteritis (AGE) due to group A rotavirus (RVA) agent is one of the major causes of hospitalization in paediatric age. The G3P[8] RVA genotype has been usually considered as one of the major human genotypes, largely circulating in Asia, but showing low detection rates in the European countries. In recent years, the G3P[8] RVAs emerged also in Europe as a predominant genotype and the viral strains detected revealed high similarities with equine-like G3P[8] RVA strains, resulting in a new variant circulating in humans and able to cause AGE in the paediatric population.

          Case presentation

          An 8-year-old boy was admitted to the Emergency Room because he had suffered from severe diarrhoea, vomiting, and high fever over the previous two days. Severe dehydration was evident based on low serum concentrations of potassium and sodium, low glycaemia, and pre-renal failure (creatinine 2.48 mg/dL, urea 133 mg/dL). Immunological tests were within normal range. Enzyme immunoassay for the detection of RV was positive, and a sample of faeces was collected in order to perform the molecular characterization of the viral strain. The phylogenetic trees revealed relatedness between the VP7 and VP4 genes of the G3P[8] RVA Italian strain (namely PG2) and those belonging to recent G3P[8] RVAs detected worldwide. The G3 VP7 belonged to the G3-I lineage and shared the highest nucleotide sequence identity (99.8%) with the equine-like G3 previously identified in other countries. The P [8] VP4 revealed a similar clustering pattern to that observed for the VP7. In addition, the molecular characterization of the 11 gene segments of strain PG2 revealed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genomic constellation.

          Conclusions

          This case shows the first detection in Italy of a reassortant G3P[8] RVA associated with a severe AGE, which is unusual in a school-age child without any known severe underlying problems. The findings reported in this paper highlight the importance of continuously monitoring the RVA strains circulating in paediatric age in order to detect novel viral variants able to spread in the general population.

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          Most cited references24

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          Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.

          The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
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            Identification of group A rotavirus gene 4 types by polymerase chain reaction.

            Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
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              Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes.

              65 episodes of rotavirus diarrhoea, detected during a longitudinal follow-up of 336 infants from birth to 24-32 months of age, were analyzed for clinical symptoms. Rotavirus gastroenteritis was characterized by watery diarrhoea, vomiting (particularly in older children), fever and dehydration. A 0-20 point numerical score was devised according to the distribution of clinical features in the patients. Using this system, the mean severity score for the 65 episodes of rotavirus diarrhoea was 11.0 +/- 3.7 as compared to 5.6 +/- 3.2 for the 183 episodes of non-rotavirus diarrhoea in the same population (p less than 0.0001, t-test). The 20 point score is proposed for analysis of efficacy studies of candidate rotavirus vaccines.
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                Author and article information

                Contributors
                +39 075 5784417 , susanna.esposito@unimi.it
                barbara.camilloni@unipg.it
                bianchini.sonia@fastwebnet.it
                giovanni.ianiro@iss.it
                ilaria.polinori@hotmail.it
                edoardo.farinelli@gmail.com
                marina.monini@iss.it
                nicola.principi@unimi.it
                Journal
                Virol J
                Virol. J
                Virology Journal
                BioMed Central (London )
                1743-422X
                15 May 2019
                15 May 2019
                2019
                : 16
                : 64
                Affiliations
                [1 ]ISNI 0000 0004 1757 3630, GRID grid.9027.c, Paediatric Clinic, Department of Surgical and Biomedical Sciences, , Università degli Studi di Perugia, ; Piazza Menghini 1, 06129 Perugia, Italy
                [2 ]ISNI 0000 0004 1757 3630, GRID grid.9027.c, Microbiology Unit, Department of Medicine, , Università degli Studi di Perugia, ; Perugia, Italy
                [3 ]ISNI 0000 0000 9120 6856, GRID grid.416651.1, Department of Food Safety, Nutrition and Veterinary Public Health, , Istituto Superiore di Sanità, ; Rome, Italy
                [4 ]ISNI 0000 0004 1757 2822, GRID grid.4708.b, Università degli Studi di Milano, ; Milan, Italy
                Author information
                http://orcid.org/0000-0003-4103-2837
                Article
                1173
                10.1186/s12985-019-1173-1
                6521491
                31092258
                6c02081f-4329-4ece-b354-0049ef269c1c
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 6 January 2019
                : 2 May 2019
                Funding
                Funded by: WAidid
                Award ID: WAidid 2018_02
                Award Recipient :
                Categories
                Case Report
                Custom metadata
                © The Author(s) 2019

                Microbiology & Virology
                acute gastroenteritis,diarrhoea,gastrointestinal virus,g3p[8] rotavirus a,rotavirus

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