Roberto Delogu

Molecular characterization of rotavirus strains from children with diarrhea in Italy, 2007-2009.

The surveillance network RotaNet‐Italia was established in 2007 in order to investigate the diversity of co‐circulating rotavirus strains in Italy, and to provide a baseline for future assessment of possible effects of vaccine implementation in selecting novel versus common rotavirus strains. A total of 2,645 rotavirus strains from pediatric patients with acute diarrhea were collected over three consecutive seasons from September 2006 through August 2009, and partially characterized by standardized multiplex RT‐PCR. Most of strains (89.1%) belonged to genotypes G1–G4, and G9, associated with either P[8] or P[4], commonly found in humans worldwide. However, in at least 2.0% of cases, viruses exhibited either a G or P type typical of animal viral strains, suggesting gene reassortment events between rotaviruses of different origin. Mixed infections with two or more rotavirus strains were observed frequently (7.6% of patients), and depended on the frequencies of co‐circulating rotaviruses of one particular genotype. The numbers and genotypes of likely natural reassortants of common genotype rotaviruses were found to be correlated with the observed numbers and genotypes of mixed infections. Large variation in the relative frequency of different rotavirus genotypes was observed between different seasons and/or areas of Italy, suggesting independent evolution or differential introduction of viral strains with respect to both time and space. J. Med. Virol. 83:1657–1668, 2011.

Rotavirus Genotypes in Sewage Treatment Plants and in Children Hospitalized with Acute Diarrhea in Italy in 2010 and 2011

ABSTRACT Although the molecular surveillance network RotaNet-Italy provides useful nationwide data on rotaviruses causing severe acute gastroenteritis in children in Italy, scarce information is available on rotavirus circulation in the general Italian population, including adults with mild or asymptomatic infection. We investigated the genotypes of rotaviruses present in urban wastewaters and compared them with those of viral strains from clinical pediatric cases. During 2010 and 2011, 285 sewage samples from 4 Italian cities were tested by reverse transcription-PCRs (RT-PCRs) specific for rotavirus VP7 and VP4 genes. Rotavirus was detected in 172 (60.4%) samples, 26 of which contained multiple rotavirus G (VP7 gene) genotypes, for a total of 198 G types. Thirty-two samples also contained multiple P (VP4 gene) genotypes, yielding 204 P types in 172 samples. Genotype G1 accounted for 65.6% of rotaviruses typed, followed by genotypes G2 (20.2%), G9 (7.6%), G4 (4.6%), G6 (1.0%), G3 (0.5%), and G26 (0.5%). VP4 genotype P[8] accounted for 75.0% of strains, genotype P[4] accounted for 23.0% of strains, and the uncommon genotypes P[6], P[9], P[14], and P[19] accounted for 2.0% of strains altogether. These rotavirus genotypes were also found in pediatric patients hospitalized in the same areas and years but in different proportions. Specifically, genotypes G2, G9, and P[4] were more prevalent in sewage samples than among samples from patients, which suggests either a larger circulation of the latter strains through the general population not requiring medical care or their greater survival in wastewaters. A high level of nucleotide identity in the G1, G2, and G6 VP7 sequences was observed between strains from the environment and those from patients.

Genetic diversity of G9P[8] rotavirus strains circulating in Italy in 2007 and 2010 as determined by whole genome sequencing

This study reports the molecular characterization of G9P[8] rotavirus strains from children with acute diarrhea identified in different cities of Italy, in 2007 and 2010. Seventeen samples exhibited a G9P[8] genotype by RT-PCR and semi-nested PCR. Preliminary sequence analysis of the VP7 and VP8(*) encoding genes revealed nucleotide identities ranging between 96% and 100%. Full genome sequencing of four G9P[8] strains selected in different cities or years showed that the investigated Italian strains possessed a complete Wa-like genotype constellation. However, phylogenetic analyses assigned strains to different clusters reflecting point mutations and possibly earlier reassortment between Wa-like RVA strains. Deduced amino acid sequence of the VP7 and VP4 genes for the G9P[8] strains revealed at least five substitutions in relevant antigenic sites of both proteins.

Unexpected spreading of G12P[8] rotavirus strains among young children in a small area of central Italy

Rotavirus gastroenteritis is associated mainly with the five genotypes G1,3,4,9P[8] and G2P[4] that are common worldwide, but emerging strains including G6, G8, and G12 are also reported sporadically. G12P[8] rotavirus was observed unexpectedly to spread in a limited area of Italy during the rotavirus surveillance season 2012–2013. All strains were genotyped for VP7 and VP4 and subjected to phylogenetic analysis. Amino acid sequences of antigenic regions were compared with vaccine and field strains. G12P[8] strains were detected in the stools of 52 of 69 (75%) children infected with rotavirus in the central Italian region of Umbria. All G12 strains belonged to lineage III, and presented the P[8] genotype. Sequence analysis showed close nucleotide identity of both VP4 and VP7 genes among Umbria G12P[8] strains. The VP7 gene was also similar to other G12 strains circulating in different years and countries, and the VP4 gene was closely related to other local and global P[8] strains possessing different G‐types. Overall findings suggest either the introduction and evolution of a G12 VP7 gene into the local Wa‐like rotavirus population or the spreading of a strain novel for the area. Comparison of the VP8* and VP7 antigenic regions showed high conservation between the amino acid sequences of Umbria G12P[8] strains, and revealed various substitutions in the VP8* antigenic regions between the Italian G12P[8] strains and RotaTeq™ and Rotarix™ vaccine strains. The sudden and unexpected emergence of G12P[8] rotavirus confirms that these strains have the potential to become a sixth common genotype across the world. J. Med. Virol. 87:1292–1302, 2015.

Molecular characterization of human G8P[4] rotavirus strains in Italy: proposal of a more complete subclassification of the G8 genotype in three major lineages.

In 2011, two children with acute rotavirus gastroenteritis were hospitalized in Sardinia, Italy. Two RVA strains with G8P[4] genotype were detected in their stools, and were named SS56/2011 and SS65/2011. The aim of the study was to characterize these two rare strains, collected within a national RVA gastroenteritis surveillance program. Eight of the 11 RVA genes were sequenced and phylogenetic analysis performed. VP7 amino acid sequence was also analyzed. Sequencing of genes encoding the VP4, VP6, VP7, and NSP1-5 proteins classified both strains as G8-P[4]-I2-A2-N2-T2-E2-H2, not detected previously in Italy. Phylogenetic analysis revealed that most genes of Italian RVA strains were closely similar to typical DS-1 like strains circulating worldwide, whereas the VP7 gene was strictly related to G8 strains firstly reported in Africa. This finding of G8P[4] RVA strains with a DS-1 like genomic constellation also in a southern European country further confirms the wide circulation of this uncommon genotype in the world. Comparison of the deduced amino acid sequence of the VP7 capsid protein of the Italian G8P[4] RVA strains with sequences reported previously suggests that the G8 genotype should be divided into three major lineages.

Detection of unusual G6 rotavirus strains in Italian children with diarrhoea during the 2011 surveillance season.

Two rare G6 rotavirus A (RVA) strains, designated as RVA/human‐wt/ITA/CEC06/2011/G6P[6] and RVA/human‐wt/ITA/PG05/2011/G6P[9], were identified in stool specimens from children hospitalized in Central Italy. After PCR genotyping, the samples CEC06 and PG05 gave G‐UD‐P[6] and G‐UD‐P[9] genotypes, respectively. To determine the G‐type and to characterize further the two strains, sequencing of 8 of the 11 genomic segments was performed. CEC06 and PG05 strains were found to possess unusual genotype constellations: G6‐P[6]‐I2‐A2‐N2‐T2‐E2‐H2 and G6‐P[9]‐I2‐A3‐N2‐T3‐E3‐H3, respectively. This study reports the first detection of rare G6P[6] and G6P[9] RVA strains in peninsular Italy. Phylogenetic analysis of VP4 (VP8*), VP7, VP6, and NSP1‐5 showed no evidence of zoonosis or inter‐species reassortment, revealing for both strains constellations previously associated to human cases. J Med. Virol. 85:1860–1869, 2013.

Full-Genome Characterization of a G8P[8] Rotavirus That Emerged among Children with Diarrhea in Croatia in 2006

ABSTRACT The whole genome of a G8P[8] rotavirus from the 2006 epidemic in Croatia was sequenced and showed a Wa-like genotype constellation. Its VP7 gene clustered with DS-1-like G8 African rotaviruses and a G8P[4] German strain. Remaining genes clustered with contemporary Belgian G1P[8] rotaviruses, suggesting reassortment between human G8 and G1P[8] rotaviruses in Croatia or other European countries.

Molecular analysis of group A rotaviruses detected in adults and adolescents with severe acute gastroenteritis in Italy in 2012

Hospital‐based surveillance of acute gastroenteritis caused by rotavirus has produced ample knowledge on the infection in children, whereas little is known on rotavirus infection among adults. The Italian surveillance program RotaNet‐Italia collected 1,595 samples from patients admitted to hospital with gastroenteritis in 2012. All patients presented with vomiting, diarrhea, fever, and/or abdominal pain. Forty‐two samples obtained by the RotaNet‐Italia (2.6%) were from adolescents or adults (10–89 years). The study compared the genotypes and gene sequences of the rotavirus strains identified in adults with strains obtained from children worldwide. All 42 Italian strains were genotyped by the EuroRotaNet RT‐nested‐PCR protocols, and 12 rotaviruses from patients >13‐year‐old were subjected to nucleotide sequencing of their VP7 and/or VP4 genes. All strains analyzed belonged to the common human genotypes G1P[8], G2P[4], G4P[8], and G9P[8], except an uncommon G3P[19] genotype detected in a single patient. Phylogenetic analysis of the 12 strains showed that within each genotype they clustered in RVA lineages reported worldwide. The amino acid sequences of the VP7 and the VP8* hypervariable regions were highly conserved between the RVA strains collected from adults and children, in each lineage. Genotyping, phylogenetic analysis, and the study of viral epitopes revealed that rotaviruses circulating in adults in Italy are closely similar to the strains circulating in children, with high nucleotide identity particularly with strains reported in Europe and Asia. The circulation of the same rotavirus strains in both children and adults suggests that adults may contribute to sustain the circulation of rotaviruses through the population. J. Med. Virol. 86:1073–1082, 2014.

Detection of Rare G3P[19] Group A Rotavirus in Human Patient, Italy

Infection with a rare G3P[19] rotavirus A strain was identified in an immunosuppressed patient in Italy. The strain showed a P[19] viral protein 4 gene and a complete AU-1–like genomic constellation. Phylogenetic analyses showed high nucleotide identity between this strain and G3P[19] rotavirus A strains from Asia, indicating possible reassortment events.

Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009.

Rotavirus strains with the uncommon genotype G10 have been detected sporadically in cases of acute gastroenteritis in humans and are thought to be transmitted zoonotically. During 2009, 10 G10P[8] rotavirus strains were detected in the stools of children hospitalized with acute diarrhoea in several paediatric hospitals in Italy. The phylogenetic analysis of the VP7 gene of the Italian G10P[8] strains analysed revealed nucleotide identities ranging from 94 to 99 %. Molecular characterization of the 11 genomic segments was performed for one of the G10 strains, which displayed a complete genomic constellation 1 for the non-G genes. The analysis of the deduced amino acid sequences of the G10 VP7 epitopes revealed low amino acid identity with common human strains of different G genotype and with the VP7 proteins included in both anti-rotavirus commercial vaccines (Rotarix and RotaTeq). Amongst the common G genotypes, the VP7 amino acid sequence of the G10 strains showed a high similarity with sequences from G9 strains. A hydrophobic cluster analysis (HCA) of the VP7 protein including aa 20-298 was performed for the G10 Italian sequences in comparison with the major human group A rotavirus G genotypes. The HCA analysis confirmed the findings obtained previously by amino acid analysis of the VP7 epitopes, detecting a genotype-specific pattern of hydrophobicity in the hypervariable regions of the major outer capsid protein.